U.S. patent number 11,034,937 [Application Number 16/414,550] was granted by the patent office on 2021-06-15 for car enzymes and improved production of fatty alcohols.
This patent grant is currently assigned to Genomatica, Inc.. The grantee listed for this patent is Genomatica, Inc.. Invention is credited to Vikranth Arlagadda, Elizabeth J. Clarke, Derek L. Greenfield, Eli S. Groban, Zhihao Hu, Sungwon Lee, Xuezhi Li, Baolong Zhu.
United States Patent |
11,034,937 |
Greenfield , et al. |
June 15, 2021 |
Car enzymes and improved production of fatty alcohols
Abstract
The disclosure relates to variant carboxylic acid reductase
(CAR) enzymes for the improved production of fatty alcohols in
recombinant host cells.
Inventors: |
Greenfield; Derek L. (South San
Francisco, CA), Clarke; Elizabeth J. (South San Francisco,
CA), Groban; Eli S. (South San Francisco, CA), Arlagadda;
Vikranth (South San Francisco, CA), Lee; Sungwon (South
San Francisco, CA), Li; Xuezhi (South San Francisco, CA),
Hu; Zhihao (South San Francisco, CA), Zhu; Baolong
(South San Francisco, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
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Assignee: |
Genomatica, Inc. (San Diego,
CA)
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Family
ID: |
1000005617071 |
Appl.
No.: |
16/414,550 |
Filed: |
May 16, 2019 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20200109374 A1 |
Apr 9, 2020 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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15695205 |
Sep 5, 2017 |
10301603 |
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15154636 |
Sep 12, 2017 |
9758769 |
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14390350 |
May 17, 2016 |
9340801 |
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PCT/US2013/035040 |
Apr 2, 2013 |
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61619309 |
Apr 2, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y
102/99006 (20130101); C12P 7/04 (20130101); C12N
9/0008 (20130101); C12P 7/6409 (20130101); Y02P
20/52 (20151101) |
Current International
Class: |
C12P
7/04 (20060101); C12N 9/02 (20060101); C12P
7/64 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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102264910 |
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Nov 2011 |
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CN |
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10 2004 052 115 |
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Apr 2006 |
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DE |
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2012-506715 |
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Mar 2012 |
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JP |
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WO-91/16427 |
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Oct 1991 |
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WO |
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WO-2007/136762 |
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Nov 2007 |
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WO |
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WO-2008/119082 |
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Oct 2008 |
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WO |
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WO-2008/147781 |
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Dec 2008 |
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WO |
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WO-2009/085278 |
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Jul 2009 |
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WO |
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WO-2010/062480 |
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Jun 2010 |
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WO |
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WO-2010/062480 |
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Jun 2010 |
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WO |
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WO-2010/127318 |
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Nov 2010 |
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WO |
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WO-2010/135624 |
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Nov 2010 |
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WO |
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WO-2011/047101 |
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Apr 2011 |
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WO |
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WO-2012/135789 |
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Oct 2012 |
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WO |
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WO-2012/154329 |
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Nov 2012 |
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WO |
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Primary Examiner: Saidha; Tekchand
Attorney, Agent or Firm: Harness, Dickey & Pierce,
P.L.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No.
15/695,205, filed Sep. 5, 2017, which is a continuation of U.S.
application Ser. No. 15/154,636, filed May 13, 2016, now U.S. Pat.
No. 9,758,769, which is a continuation of U.S. application Ser. No.
14/390,350, filed Oct. 2, 2014, now U.S. Pat. No. 9,340,801, which
is the U.S. National Stage of International Application No.
PCT/US2013/035040, filed Apr. 2, 2013, which claims the benefit of
U.S. Provisional Application No. 61/619,309, filed Apr. 2, 2012,
the entire disclosures of which are hereby incorporated by
reference.
Claims
What is claimed is:
1. A variant carboxylic acid reductase (CAR) polypeptide comprising
an amino acid sequence having at least 85% sequence identity to the
wild-type CAR polypeptide of SEQ ID NO: 7, wherein the variant CAR
polypeptide is genetically engineered to have at least one mutation
at an amino acid position selected from the group consisting of
E636K; L1128S; M259I; T396S; A574T; T90M; E20F; E20L; E20Y; V191A;
Q473A; Q473F; Q473K; Q473M; Q473R; Q473V; V926G; S927I; S927V;
L1128A; L1128G; L1128K; L1128M; L1128P; L1128R; L1128T; L1128V;
L1128Y; D18V and D292N; E20K and V191A; P809L and M1062V; D143E and
A612T; M1062T and R1080H; E936K, and P1134R; I870V, S927I, S985I,
and I1164F; L799M, V810F, S927R, M1062L, A1158V, and F1170I; and
L80Q, T231M, F288L, A418T, V530M, A541V, G677D, and P712A, wherein
expression of the variant CAR polypeptide in a recombinant host
cell results in a higher titer of fatty alcohol compositions
compared to a recombinant host cell expressing the wild type CAR
polypeptide of SEQ ID NO:7.
2. The variant CAR polypeptide of claim 1, wherein said variant
polypeptide comprises: (a) the mutations P809L and M1062V; (b) the
mutations D143E and A612T; (c) the mutations M1062T and R1080H; (d)
the mutations E936K, and P1134R; (e) the mutations I870V, S927I,
S985I, and I1164F; (f) the mutations L799M, V810F, S927R, M1062L,
A1158V, and F1170I; or (g) the mutations L80Q, T231M, F288L, A418T,
V530M, A541V, G677D, and P712A.
3. A recombinant host cell comprising a polynucleotide sequence
encoding a variant carboxylic acid reductase (CAR) polypeptide
having at least 85% sequence identity to the wild-type CAR
polypeptide of SEQ ID NO: 7 and having at least one mutation at an
amino acid position selected from the group consisting of E636K;
L1128S; M259I; T396S; A574T; T90M; E20F; E20L; E20Y; V191A; Q473A;
Q473F; Q473K; Q473M; Q473R; Q473V; V926G; S927I; S927V; L1128A;
L1128G; L1128K; L1128M; L1128P; L1128R; L1128T; L1128V; L1128Y;
D18V and D292N; E20K and V191A; P809L and M1062V; D143E and A612T;
M1062T and R1080H; E936K, and P1134R; I870V, S927I, S985I, and
I1164F; L799M, V810F, S927R, M1062L, A1158V, and F1170I; and L80Q,
T231M, F288L, A418T, V530M, A541V, G677D, and P712A, wherein the
recombinant host cell produces a fatty alcohol composition at a
higher titer or yield than a host cell expressing the wild type CAR
polypeptide of SEQ ID NO: 7 when cultured in a medium containing a
carbon source under conditions effective to express said variant
CAR polypeptide.
4. The recombinant host cell of claim 3, further comprising a
polynucleotide encoding a thioesterase polypeptide.
5. The recombinant host cell of claim 4, further comprising a
polynucleotide encoding (i) a FabB polypeptide and a FadR
polypeptide, or (ii) a fatty aldehyde reductase.
6. The recombinant host cell according to claim 3, wherein said
recombinant host cell has a titer or a yield that is at least 3
times greater than the titer or the yield of a host cell expressing
the corresponding wild type CAR polypeptide when cultured under the
same conditions as the recombinant host cell.
7. The recombinant host cell of claim 6, wherein said recombinant
host cell has a titer of from 30 g/L to 250 g/L, or a titer of from
90 g/L to 120 g/L.
8. The recombinant host cell of claim 6, wherein said recombinant
host cell has a yield from 10% to 40%.
9. A cell culture comprising the recombinant host cell of claim
3.
10. The cell culture of claim 9, wherein said cell culture has
productivity that is at least 3 times greater than the productivity
of a cell culture that expresses the corresponding wild type CAR
polypeptide.
11. The cell culture of claim 10, wherein said productivity ranges
from 0.7 mg/L/hr to 3 g/L/hr.
12. The cell culture of claim 11, wherein the culture medium
comprises a fatty alcohol composition.
13. The recombinant host cell according to claim 3, wherein the
fatty alcohol composition collects in an organic phase
extracellularly.
14. The recombinant host cell of claim 13, wherein the fatty
alcohol composition comprises one or more of a C6, C8, C10, C12,
C13, C14, C15, C16, C17, or C18 fatty alcohol.
15. The recombinant host cell of claim 13, wherein the fatty
alcohol composition comprises a C10:1, C12:1, C14:1, C16:1, or a
C18:1 unsaturated fatty alcohol.
16. The recombinant host cell of claim 13, wherein the fatty
alcohol composition comprises C12 and C14 fatty alcohols.
17. The recombinant host cell of claim 16, wherein the fatty
alcohol composition comprises C12 and C14 fatty alcohols at a ratio
of 3:1.
18. The recombinant host cell of claim 13, wherein the fatty
alcohol composition comprises unsaturated fatty alcohols or
saturated fatty alcohols.
19. The recombinant host cell of claim 18, wherein the fatty
alcohol composition comprises an unsaturated fatty alcohol having a
double bond at position 7 in the carbon chain between C7 and C8
from the reduced end of the fatty alcohol.
20. The recombinant host cell of claim 13, wherein the fatty
alcohol composition comprises branched chain fatty alcohols.
21. A method of making a fatty alcohol composition at a high titer,
yield or productivity, comprising the steps of: (a) engineering a
recombinant host cell according to claim 3; (b) culturing said
recombinant host cell in a medium comprising a carbon source; and
(c) isolating said fatty alcohol composition from said medium.
Description
SEQUENCE LISTING
The instant application contains a Sequence Listing, which has been
submitted in ASCII format via EFS-Web and is hereby incorporated by
reference in its entirety. The ASCII copy, created on Apr. 2, 2013,
is named LS00039PCT_SL.txt and is 89,038 bytes in size.
FIELD OF THE DISCLOSURE
The disclosure relates to variant carboxylic acid reductase (CAR)
enzymes for the improved production of fatty alcohols in
recombinant host cells. The disclosure further relates to variant
CAR nucleic acids and polypeptides as well as recombinant host
cells and cell cultures. Further encompassed are methods of making
fatty alcohol compositions.
BACKGROUND OF THE DISCLOSURE
Fatty alcohols make up an important category of industrial
biochemicals. These molecules and their derivatives have numerous
uses, including as surfactants, lubricants, plasticizers, solvents,
emulsifiers, emollients, thickeners, flavors, fragrances, and
fuels. In industry, fatty alcohols are produced via catalytic
hydrogenation of fatty acids produced from natural sources, such as
coconut oil, palm oil, palm kernel oil, tallow and lard, or by
chemical hydration of alpha-olefins produced from petrochemical
feedstock. Fatty alcohols derived from natural sources have varying
chain lengths. The chain length of fatty alcohols is important with
respect to particular applications. In nature, fatty alcohols are
also made by enzymes that are able to reduce acyl-ACP or acyl-CoA
molecules to the corresponding primary alcohols (see, for example,
U.S. Patent Publication Nos. 20100105955, 20100105963, and
20110250663, which are incorporated by reference herein).
Current technologies for producing fatty alcohols involve inorganic
catalyst-mediated reduction of fatty acids to the corresponding
primary alcohols, which is costly, time consuming and cumbersome.
The fatty acids used in this process are derived from natural
sources (e.g., plant and animal oils and fats, supra). Dehydration
of fatty alcohols to alpha-olefins can also be accomplished by
chemical catalysis. However, this technique is nonrenewable and
associated with high operating cost and environmentally hazardous
chemical wastes. Thus, there is a need for improved methods for
fatty alcohol production and the instant disclosure addresses this
need.
SUMMARY
One aspect of the disclosure provides a variant carboxylic acid
reductase (CAR) polypeptide comprising an amino acid sequence
having at least about 90% sequence identity to SEQ ID NO: 7,
wherein the variant CAR polypeptide is genetically engineered to
have at least one mutation at an amino acid position selected from
the group of amino acid positions 3, 18, 20, 22, 80, 87, 191, 288,
473, 535, 750, 827, 870, 873, 926, 927, 930, and 1128. Herein, the
expression of the variant CAR polypeptide in a recombinant host
cell results in a higher titer of fatty alcohol compositions
compared to a recombinant host cell expressing a corresponding wild
type polypeptide. In a related aspect, the CAR polypeptide is a
CarB polypeptide. In another related aspect, the variant CAR
polypeptide comprises a mutation at positions S3R, D18R, D18L,
D18T, D18P, E20V, E205, E20R, S22R, S22N, S22G, L80R, R87G, R87E,
V191S, F288R, F288S, F288G, Q473L, Q473W, Q473Y, Q473I, Q473H,
A535S, D750A, R827C, R827A, I870L, R873S, V926A, V926E, S927K,
S927G, M930K, M930R and/or L1128W. In a related aspect, the CAR
polypeptide includes mutation A535S; or mutations E20R, F288G,
Q473I and A535S; or mutations E20R, F288G, Q473H, A535S, R827A and
S927G; or mutations E20R, S22R, F288G, Q473H, A535S, R827A and
S927G; or mutations S3R, E20R, S22R, F288G, Q473H, A535S, R873S,
S927G, M930R and L1128W; or E20R, S22R, F288G, Q473H, A535S, R873S,
S927G, M930R and L1128W; or mutations D18R, E20R, S22R, F288G,
Q473I, A535S, S927G, M930K and L1128W; or mutations E20R, S22R,
F288G, Q473I, A535S, R827C, V926E, S927K and M930R; or mutations
D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K and L1128W; or
mutations E20R, S22R, F288G, Q473H, A535S, R827C, V926A, S927K and
M930R; or mutations E20R, S22R, F288G, Q473H, A535S and R827C; or
mutations E20R, S22R, F288G, Q473I, A535S, R827C and M930R; or
mutations E20R, S22R, F288G, Q473I, A535S, I870L, S927G and M930R;
or mutations E20R, S22R, F288G, Q473I, A535S, I870L and S927G; or
mutations D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L,
V926A and S927G; or mutations E20R, S22R, F288G, Q473H, A535S,
R827C, I870L and L1128W; or mutations D18R, E20R, S22R, F288G,
Q473H, A535S, R827C, I870L, S927G and L1128W; or mutations E20R,
S22R, F288G, Q473I, A535S, R827C, I870L, S927G and L1128W; or
mutations E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G,
M930K and L1128W; or mutations E20R, S22R, F288G, Q473H, A535S,
I870L, S927G and M930K; or mutations E20R, F288G, Q473I, A535S,
I870L, M930K; or mutations E20R, S22R, F288G, Q473H, A535S, S927G,
M930K and L1128W; or mutations D18R, E20R, S22R, F288G, Q473I,
A535S, S927G and L1128W; or mutations E20R, S22R, F288G, Q473I,
A535S, R827C, I870L and S927G; or mutations D18R, E20R, S22R,
F288G, Q473I, A535S, R827C, I870L, S927G and L1128W; or mutations
D18R, E20R, S22R, F288G, Q473I, A535S, S927G, M930R and L1128W; or
mutations E20R, S22R, F288G, Q473H, A535S, V926E, S927G and M930R;
or mutations E20R, S22R, F288G, Q473H, A535S, R827C, I870L, V926A
and L1128W; or combinations thereof.
Another aspect of the disclosure provides a host cell including a
polynucleotide sequence encoding a variant carboxylic acid
reductase (CAR) polypeptide having at least 90% sequence identity
to SEQ ID NO: 7 and having at least one mutation at an amino acid
position including amino acid positions 3, 18, 20, 22, 80, 87, 191,
288, 473, 535, 750, 827, 870, 873, 926, 927, 930, and 1128, wherein
the genetically engineered host cell produces a fatty alcohol
composition at a higher titer or yield than a host cell expressing
a corresponding wild type CAR polypeptide when cultured in a medium
containing a carbon source under conditions effective to express
the variant CAR polypeptide, and wherein the SEQ ID NO: 7 is the
corresponding wild type CAR polypeptide. In a related aspect, the
recombinant host cell further includes a polynucleotide encoding a
thioesterase polypeptide. In another related aspect, the
recombinant host cell further includes a polynucleotide encoding a
FabB polypeptide and a FadR polypeptide. In another related aspect,
the disclosure provides a recombinant host cell that includes a
polynucleotide encoding a fatty aldehyde reductase (AlrA) and a
cell culture containing it.
Another aspect of the disclosure provides a recombinant host cell,
wherein the genetically engineered host cell has a titer that is at
least 3 times greater than the titer of a host cell expressing the
corresponding wild type CAR polypeptide when cultured under the
same conditions as the genetically engineered host cell. In one
related aspect, the genetically engineered host cell has a titer of
from about 30 g/L to about 250 g/L. In another related aspect, the
genetically engineered host cell has a titer of from about 90 g/L
to about 120 g/L.
Another aspect of the disclosure provides a recombinant host cell,
wherein the genetically engineered host cell has a yield that is at
least 3 times greater than the yield of a host cell expressing the
corresponding wild type CAR polypeptide when cultured under the
same conditions as the genetically engineered host cell. In one
related aspect, the genetically engineered host cell has a yield
from about 10% to about 40%.
The disclosure further encompasses a cell culture including the
recombinant host cell as described herein. In a related aspect, the
cell culture has a productivity that is at least about 3 times
greater than the productivity of a cell culture that expresses the
corresponding wild type CAR polypeptide. In another related aspect,
the productivity ranges from about 0.7 mg/L/hr to about 3 g/L/hr.
In another related aspect, the culture medium comprises a fatty
alcohol composition. The fatty alcohol composition is produced
extracellularly. The fatty alcohol composition may include one or
more of a C6, C8, C10, C12, C13, C14, C15, C16, C17, or C18 fatty
alcohol; or a C10:1, C12:1, C14:1, C16:1, or a C18:1 unsaturated
fatty alcohol. In another related aspect, the fatty alcohol
composition comprises C12 and C14 fatty alcohols. In yet, another
related aspect, the fatty alcohol composition comprises C12 and C14
fatty alcohols at a ratio of about 3:1. In still another related
aspect, the fatty alcohol composition encompasses unsaturated fatty
alcohols. In addition, the fatty alcohol composition may include a
fatty alcohol having a double bond at position 7 in the carbon
chain between C7 and C8 from the reduced end of the fatty alcohol.
In another aspect, the fatty alcohol composition includes saturated
fatty alcohols. In another aspect, the fatty alcohol composition
includes branched chain fatty alcohols.
The disclosure further contemplates a method of making a fatty
alcohol composition at a high titer, yield or productivity,
including the steps of engineering a recombinant host cell;
culturing the recombinant host cell in a medium including a carbon
source; and optionally isolating the fatty alcohol composition from
the medium
BRIEF DESCRIPTION OF THE DRAWINGS
The present disclosure is best understood when read in conjunction
with the accompanying figures, which serve to illustrate the
preferred embodiments. It is understood, however, that the
disclosure is not limited to the specific embodiments disclosed in
the figures.
FIG. 1 is a schematic overview of an exemplary biosynthetic pathway
for use in production of acyl CoA as a precursor to fatty acid
derivatives in a recombinant host cell. The cycle is initiated by
condensation of malonyl-ACP and acetyl-CoA.
FIG. 2 is a schematic overview of an exemplary fatty acid
biosynthetic cycle, where malonyl-ACP is produced by the
transacylation of malonyl-CoA to malonyl-ACP (catalyzed by
malonyl-CoA:ACP transacylase; fabD), then .beta.-ketoacyl-ACP
synthase III (fabH) initiates condensation of malonyl-ACP with
acetyl-CoA. Elongation cycles begin with the condensation of
malonyl-ACP and an acyl-ACP catalyzed by .beta.-ketoacyl-ACP
synthase I (fabB) and .beta.-ketoacyl-ACP synthase II (fabF) to
produce a .beta.-keto-acyl-ACP, then the .beta.-keto-acyl-ACP is
reduced by a NADPH-dependent .beta.-ketoacyl-ACP reductase (fabG)
to produce a .beta.-hydroxy-acyl-ACP, which is dehydrated to a
trans-2-enoyl-acyl-ACP by .beta.-hydroxyacyl-ACP dehydratase (fabA
or fabZ). FabA can also isomerize trans-2-enoyl-acyl-ACP to
cis-3-enoyl-acyl-ACP, which can bypass fabI and can used by fabB
(typically for up to an aliphatic chain length of C16) to produce
.beta.-keto-acyl-ACP. The final step in each cycle is catalyzed by
a NADH or NADHPH-dependent enoyl-ACP reductase (fabI) that converts
trans-2-enoyl-acyl-ACP to acyl-ACP. In the methods described
herein, termination of fatty acid synthesis occurs by thioesterase
removal of the acyl group from acyl-ACP to release free fatty acids
(FFA). Thioesterases (e.g., tesA) hydrolyze thioester bonds, which
occur between acyl chains and ACP through sulfhydryl bonds.
FIG. 3 illustrates the structure and function of the acetyl-CoA
carboxylase (accABCD) enzyme complex. Biotin carboxylase is encoded
by the accC gene, whereas biotin carboxyl carrier protein (BCCP) is
encoded by the accB gene. The two subunits involved in
carboxyltransferase activity are encoded by the accA and accD
genes. The covalently bound biotin of BCCP carries the carboxylate
moiety. The birA gene (not shown) biotinylates holo-accB.
FIG. 4 presents a schematic overview of an exemplary biosynthetic
pathway for production of fatty alcohol starting with acyl-ACP,
where the production of fatty aldehyde is catalyzed by the
enzymatic activity of acyl-ACP reductase (AAR) or thioesterase and
carboxylic acid reductase (Car). The fatty aldehyde is converted to
fatty alcohol by aldehyde reductase (also referred to as alcohol
dehydrogenase). This pathway does not include fatty acyl CoA
synthetase (fadD).
FIG. 5 illustrates fatty acid derivative (Total Fatty Species)
production by the MG1655 E. coli strain with the fadE gene
attenuated (i.e., deleted) compared to fatty acid derivative
production by E. coli MG1655. The data presented in FIG. 5 shows
that attenuation of the fadE gene did not affect fatty acid
derivative production.
FIGS. 6A and 6B show data for production of "Total Fatty Species"
from duplicate plate screens when plasmid pCL-WT TRC WT TesA was
transformed into each of the strains shown in the figures and a
fermentation was run in FA2 media with 20 hours from induction to
harvest at both 32.degree. C. (FIG. 6A) and 37.degree. C. (FIG.
6B).
FIGS. 7A and 7B provide a diagrammatic depiction of the iFAB138
locus, including a diagram of cat-loxP-T5 promoter integrated in
front of FAB138 (7A); and a diagram of iT5_138 (7B). The sequence
of cat-loxP-T5 promoter integrated in front of FAB138 with 50 base
pair of homology shown on each side of cat-loxP-T5 promoter region
is provided as SEQ ID NO:1 and the sequence of the iT5_138 promoter
region with 50 base pair homology on each side is provided as SEQ
ID NO: 2.
FIG. 8 shows the effect of correcting the rph and ilvG genes. EG149
(rph- ilvg-) and V668 (EG149 rph+ ilvG+) were transformed with
pCL-tesA (a pCL1920 plasmid containing P.sub.TRC-'tesA) obtained
from D191. The figure shows that correcting the rph and ilvG genes
in the EG149 strain allows for a higher level of FFA production
than in the V668 strain where the rph and ilvG genes were not
corrected.
FIG. 9 is a diagrammatic depiction of a transposon cassette
insertion in the yijP gene of strain LC535 (transposon hit 68F11).
Promoters internal to the transposon cassette are shown, and may
have effects on adjacent gene expression.
FIG. 10 shows conversion of free fatty acids to fatty alcohols by
CarB60 in strain V324. The figures shows that cells expressing
CarB60 from the chromosome (dark bars) convert a greater fraction
of C12 and C14 free fatty acids into fatty alcohol compared to CarB
(light bars).
FIG. 11 shows that cells expressing CarB60 from the chromosome
convert a greater fraction of C12 and C14 free fatty acids into
fatty alcohol compared to CarB.
FIG. 12 shows fatty alcohol production following fermentation of
combination library mutants.
FIG. 13 shows fatty alcohol production by carB variants in
production plasmid (carB1 and CarB2) following shake-flask
fermentation.
FIG. 14 shows fatty alcohol production by single-copy integrated
carB variants (icarB1 icarB2, icarB3, and icarB4) following
shake-flask fermentation.
FIG. 15 shows results of dual-plasmid screening system for improved
CarB variants as validated by shake-flask fermentation.
FIG. 16 shows novel CarB variants for improved production of fatty
alcohols in bioreactors.
DETAILED DESCRIPTION
General Overview
The present disclosure provides novel variant carboxylic acid
reductase (CAR) enzymes as well as their nucleic acid and protein
sequences. Further encompassed by the disclosure are recombinant
host cells and cell cultures that include the variant CAR enzymes
for the production of fatty alcohols. In order for the production
of fatty alcohols from fermentable sugars or biomass to be
commercially viable, the process must be optimized for efficient
conversion and recovery of product. The present disclosure
addresses this need by providing compositions and methods for
improved production of fatty alcohols using engineered variant
enzymes and engineered recombinant host cells. The host cells serve
as biocatalysts resulting in high-titer production of fatty
alcohols using fermentation processes. As such, the disclosure
further provides methods to create photosynthetic and heterotrophic
host cells that produce fatty alcohols and alpha-olefins of
specific chain lengths directly such that catalytic conversion of
purified fatty acids is not necessary. This new method provides
product quality and cost advantages.
More specifically, the production of a desired fatty alcohol
composition may be enhanced by modifying the expression of one or
more genes involved in a biosynthetic pathway for fatty alcohol
production, degradation and/or secretion. The disclosure provides
recombinant host cells, which have been engineered to provide
enhanced fatty alcohol biosynthesis relative to non-engineered or
native host cells (e.g., strain improvements). The disclosure also
provides polynucleotides useful in the recombinant host cells,
methods, and compositions of the disclosure. However it will be
recognized that absolute sequence identity to such polynucleotides
is not necessary. For example, changes in a particular
polynucleotide sequence can be made and the encoded polypeptide
evaluated for activity. Such changes typically comprise
conservative mutations and silent mutations (e.g., codon
optimization). Modified or mutated polynucleotides (i.e., mutants)
and encoded variant polypeptides can be screened for a desired
function, such as, an improved function compared to the parent
polypeptide, including but not limited to increased catalytic
activity, increased stability, or decreased inhibition (e.g.,
decreased feedback inhibition), using methods known in the art.
The disclosure identifies enzymatic activities involved in various
steps (i.e., reactions) of the fatty acid biosynthetic pathways
described herein according to Enzyme Classification (EC) number,
and provides exemplary polypeptides (i.e., enzymes) categorized by
such EC numbers, and exemplary polynucleotides encoding such
polypeptides. Such exemplary polypeptides and polynucleotides,
which are identified herein by Accession Numbers and/or Sequence
Identifier Numbers (SEQ ID NOs), are useful for engineering fatty
acid pathways in parental host cells to obtain the recombinant host
cells described herein. It is to be understood, however, that
polypeptides and polynucleotides described herein are exemplary and
non-limiting. The sequences of homologues of exemplary polypeptides
described herein are available to those of skill in the art using
databases (e.g., the Entrez databases provided by the National
Center for Biotechnology Information (NCBI), the ExPasy databases
provided by the Swiss Institute of Bioinformatics, the BRENDA
database provided by the Technical University of Braunschweig, and
the KEGG database provided by the Bioinformatics Center of Kyoto
University and University of Tokyo, all which are available on the
World Wide Web).
A variety of host cells can be modified to contain a fatty alcohol
biosynthetic enzymes such as those described herein, resulting in
recombinant host cells suitable for the production of fatty alcohol
compositions. It is understood that a variety of cells can provide
sources of genetic material, including polynucleotide sequences
that encode polypeptides suitable for use in a recombinant host
cell provided herein.
Definitions
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the disclosure pertains.
Although other methods and materials similar, or equivalent, to
those described herein can be used in the practice of the present
disclosure, the preferred materials and methods are described
herein. In describing and claiming the present disclosure, the
following terminology will be used in accordance with the
definitions set out below.
Accession Numbers: Sequence Accession numbers throughout this
description were obtained from databases provided by the NCBI
(National Center for Biotechnology Information) maintained by the
National Institutes of Health, U.S.A. (which are identified herein
as "NCBI Accession Numbers" or alternatively as "GenBank Accession
Numbers"), and from the UniProt Knowledgebase (UniProtKB) and
Swiss-Prot databases provided by the Swiss Institute of
Bioinformatics (which are identified herein as "UniProtKB Accession
Numbers").
Enzyme Classification (EC) Numbers: EC numbers are established by
the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (IUBMB), description of which is
available on the IUBMB Enzyme Nomenclature website on the World
Wide Web. EC numbers classify enzymes according to the reaction
catalyzed.
As used herein, the term "nucleotide" refers to a monomeric unit of
a polynucleotide that consists of a heterocyclic base, a sugar, and
one or more phosphate groups. The naturally occurring bases
(guanine, (G), adenine, (A), cytosine, (C), thymine, (T), and
uracil (U)) are typically derivatives of purine or pyrimidine,
though it should be understood that naturally and non-naturally
occurring base analogs are also included. The naturally occurring
sugar is the pentose (five-carbon sugar) deoxyribose (which forms
DNA) or ribose (which forms RNA), though it should be understood
that naturally and non-naturally occurring sugar analogs are also
included. Nucleic acids are typically linked via phosphate bonds to
form nucleic acids or polynucleotides, though many other linkages
are known in the art (e.g., phosphorothioates, boranophosphates,
and the like).
As used herein, the term "polynucleotide" refers to a polymer of
ribonucleotides (RNA) or deoxyribonucleotides (DNA), which can be
single-stranded or double-stranded and which can contain
non-natural or altered nucleotides. The terms "polynucleotide,"
"nucleic acid sequence," and "nucleotide sequence" are used
interchangeably herein to refer to a polymeric form of nucleotides
of any length, either RNA or DNA. These terms refer to the primary
structure of the molecule, and thus include double- and
single-stranded DNA, and double- and single-stranded RNA. The terms
include, as equivalents, analogs of either RNA or DNA made from
nucleotide analogs and modified polynucleotides such as, though not
limited to methylated and/or capped polynucleotides. The
polynucleotide can be in any form, including but not limited to,
plasmid, viral, chromosomal, EST, cDNA, mRNA, and rRNA.
As used herein, the terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues. The
term "recombinant polypeptide" refers to a polypeptide that is
produced by recombinant techniques, wherein generally DNA or RNA
encoding the expressed protein is inserted into a suitable
expression vector that is in turn used to transform a host cell to
produce the polypeptide.
As used herein, the terms "homolog," and "homologous" refer to a
polynucleotide or a polypeptide comprising a sequence that is at
least about 50% identical to the corresponding polynucleotide or
polypeptide sequence. Preferably homologous polynucleotides or
polypeptides have polynucleotide sequences or amino acid sequences
that have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least
about 99% homology to the corresponding amino acid sequence or
polynucleotide sequence. As used herein the terms sequence
"homology" and sequence "identity" are used interchangeably.
One of ordinary skill in the art is well aware of methods to
determine homology between two or more sequences. Briefly,
calculations of "homology" between two sequences can be performed
as follows. The sequences are aligned for optimal comparison
purposes (e.g., gaps can be introduced in one or both of a first
and a second amino acid or nucleic acid sequence for optimal
alignment and non-homologous sequences can be disregarded for
comparison purposes). In a preferred embodiment, the length of a
first sequence that is aligned for comparison purposes is at least
about 30%, preferably at least about 40%, more preferably at least
about 50%, even more preferably at least about 60%, and even more
preferably at least about 70%, at least about 80%, at least about
90%, or about 100% of the length of a second sequence. The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions of the first and second sequences are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position. The percent homology between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps and the length of
each gap, that need to be introduced for optimal alignment of the
two sequences.
The comparison of sequences and determination of percent homology
between two sequences can be accomplished using a mathematical
algorithm, such as BLAST (Altschul et al., J. Mol. Biol., 215(3):
403-410 (1990)). The percent homology between two amino acid
sequences also can be determined using the Needleman and Wunsch
algorithm that has been incorporated into the GAP program in the
GCG software package, using either a Blossum 62 matrix or a PAM250
matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length
weight of 1, 2, 3, 4, 5, or 6 (Needleman and Wunsch, J. Mol. Biol.,
48: 444-453 (1970)). The percent homology between two nucleotide
sequences also can be determined using the GAP program in the GCG
software package, using a NWSgapdna.CMP matrix and a gap weight of
40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
One of ordinary skill in the art can perform initial homology
calculations and adjust the algorithm parameters accordingly. A
preferred set of parameters (and the one that should be used if a
practitioner is uncertain about which parameters should be applied
to determine if a molecule is within a homology limitation of the
claims) are a Blossum 62 scoring matrix with a gap penalty of 12, a
gap extend penalty of 4, and a frameshift gap penalty of 5.
Additional methods of sequence alignment are known in the
biotechnology arts (see, e.g., Rosenberg, BMC Bioinformatics, 6:
278 (2005); Altschul, et al., FEBS J., 272(20): 5101-5109
(2005)).
As used herein, the term "hybridizes under low stringency, medium
stringency, high stringency, or very high stringency conditions"
describes conditions for hybridization and washing. Guidance for
performing hybridization reactions can be found in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that
reference and either method can be used. Specific hybridization
conditions referred to herein are as follows: 1) low stringency
hybridization conditions--6.times. sodium chloride/sodium citrate
(SSC) at about 45.degree. C., followed by two washes in
0.2.times.SSC, 0.1% SDS at least at 50.degree. C. (the temperature
of the washes can be increased to 55.degree. C. for low stringency
conditions); 2) medium stringency hybridization
conditions--6.times. SSC at about 45.degree. C., followed by one or
more washes in 0.2.times.SSC, 0.1% SDS at 60.degree. C.; 3) high
stringency hybridization conditions--6.times.SSC at about
45.degree. C., followed by one or more washes in 0.2..times.SSC,
0.1% SDS at 65.degree. C.; and 4) very high stringency
hybridization conditions--0.5M sodium phosphate, 7% SDS at
65.degree. C., followed by one or more washes at 0.2.times.SSC, 1%
SDS at 65.degree. C. Very high stringency conditions (4) are the
preferred conditions unless otherwise specified.
An "endogenous" polypeptide refers to a polypeptide encoded by the
genome of the parental microbial cell (also termed "host cell")
from which the recombinant cell is engineered (or "derived").
An "exogenous" polypeptide refers to a polypeptide, which is not
encoded by the genome of the parental microbial cell. A variant
(i.e., mutant) polypeptide is an example of an exogenous
polypeptide.
The term "heterologous" generally means derived from a different
species or derived from a different organism. As used herein it
refers to a nucleotide sequence or a polypeptide sequence that is
not naturally present in a particular organism. Heterologous
expression means that a protein or polypeptide is experimentally
added to a cell that does not normally express that protein. As
such, heterologous refers to the fact that a transferred protein
was initially derived from a different cell type or a different
species then the recipient. For example, a polynucleotide sequence
endogenous to a plant cell can be introduced into a bacterial host
cell by recombinant methods, and the plant polynucleotide is then a
heterologous polynucleotide in a recombinant bacterial host
cell.
As used herein, the term "fragment" of a polypeptide refers to a
shorter portion of a full-length polypeptide or protein ranging in
size from four amino acid residues to the entire amino acid
sequence minus one amino acid residue. In certain embodiments of
the disclosure, a fragment refers to the entire amino acid sequence
of a domain of a polypeptide or protein (e.g., a substrate binding
domain or a catalytic domain).
As used herein, the term "mutagenesis" refers to a process by which
the genetic information of an organism is changed in a stable
manner. Mutagenesis of a protein coding nucleic acid sequence
produces a mutant protein. Mutagenesis also refers to changes in
non-coding nucleic acid sequences that result in modified protein
activity.
As used herein, the term "gene" refers to nucleic acid sequences
encoding either an RNA product or a protein product, as well as
operably-linked nucleic acid sequences affecting the expression of
the RNA or protein (e.g., such sequences include but are not
limited to promoter or enhancer sequences) or operably-linked
nucleic acid sequences encoding sequences that affect the
expression of the RNA or protein (e.g., such sequences include but
are not limited to ribosome binding sites or translational control
sequences).
Expression control sequences are known in the art and include, for
example, promoters, enhancers, polyadenylation signals,
transcription terminators, internal ribosome entry sites (IRES),
and the like, that provide for the expression of the polynucleotide
sequence in a host cell. Expression control sequences interact
specifically with cellular proteins involved in transcription
(Maniatis et al., Science, 236: 1237-1245 (1987)). Exemplary
expression control sequences are described in, for example,
Goeddel, Gene Expression Technology: Methods in Enzymology, Vol.
185, Academic Press, San Diego, Calif. (1990).
In the methods of the disclosure, an expression control sequence is
operably linked to a polynucleotide sequence. By "operably linked"
is meant that a polynucleotide sequence and an expression control
sequence(s) are connected in such a way as to permit gene
expression when the appropriate molecules (e.g., transcriptional
activator proteins) are bound to the expression control
sequence(s). Operably linked promoters are located upstream of the
selected polynucleotide sequence in terms of the direction of
transcription and translation. Operably linked enhancers can be
located upstream, within, or downstream of the selected
polynucleotide.
As used herein, the term "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid, i.e., a
polynucleotide sequence, to which it has been linked. One type of
useful vector is an episome (i.e., a nucleic acid capable of
extra-chromosomal replication). Useful vectors are those capable of
autonomous replication and/or expression of nucleic acids to which
they are linked. Vectors capable of directing the expression of
genes to which they are operatively linked are referred to herein
as "expression vectors." In general, expression vectors of utility
in recombinant DNA techniques are often in the form of "plasmids,"
which refer generally to circular double stranded DNA loops that,
in their vector form, are not bound to the chromosome. The terms
"plasmid" and "vector" are used interchangeably herein, inasmuch as
a plasmid is the most commonly used form of vector. However, also
included are such other forms of expression vectors that serve
equivalent functions and that become known in the art subsequently
hereto. In some embodiments, the recombinant vector comprises at
least one sequence including (a) an expression control sequence
operatively coupled to the polynucleotide sequence; (b) a selection
marker operatively coupled to the polynucleotide sequence; (c) a
marker sequence operatively coupled to the polynucleotide sequence;
(d) a purification moiety operatively coupled to the polynucleotide
sequence; (e) a secretion sequence operatively coupled to the
polynucleotide sequence; and (f) a targeting sequence operatively
coupled to the polynucleotide sequence. The expression vectors
described herein include a polynucleotide sequence described herein
in a form suitable for expression of the polynucleotide sequence in
a host cell. It will be appreciated by those skilled in the art
that the design of the expression vector can depend on such factors
as the choice of the host cell to be transformed, the level of
expression of polypeptide desired, etc. The expression vectors
described herein can be introduced into host cells to produce
polypeptides, including fusion polypeptides, encoded by the
polynucleotide sequences as described herein.
Expression of genes encoding polypeptides in prokaryotes, for
example, E. coli, is most often carried out with vectors containing
constitutive or inducible promoters directing the expression of
either fusion or non-fusion polypeptides. Fusion vectors add a
number of amino acids to a polypeptide encoded therein, usually to
the amino- or carboxy-terminus of the recombinant polypeptide. Such
fusion vectors typically serve one or more of the following three
purposes: (1) to increase expression of the recombinant
polypeptide; (2) to increase the solubility of the recombinant
polypeptide; and (3) to aid in the purification of the recombinant
polypeptide by acting as a ligand in affinity purification. Often,
in fusion expression vectors, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
polypeptide. This enables separation of the recombinant polypeptide
from the fusion moiety after purification of the fusion
polypeptide. In certain embodiments, a polynucleotide sequence of
the disclosure is operably linked to a promoter derived from
bacteriophage T5. In certain embodiments, the host cell is a yeast
cell, and the expression vector is a yeast expression vector.
Examples of vectors for expression in yeast S. cerevisiae include
pYepSec1 (Baldari et al., EMBO J., 6: 229-234 (1987)), pMFa (Kurjan
et al., Cell, 30: 933-943 (1982)), pJRY88 (Schultz et al., Gene,
54: 113-123 (1987)), pYES2 (Invitrogen Corp., San Diego, Calif.),
and picZ (Invitrogen Corp., San Diego, Calif.). In other
embodiments, the host cell is an insect cell, and the expression
vector is a baculovirus expression vector. Baculovirus vectors
available for expression of proteins in cultured insect cells
(e.g., Sf9 cells) include, for example, the pAc series (Smith et
al., Mol. Cell Biol., 3: 2156-2165 (1983)) and the pVL series
(Lucklow et al., Virology, 170: 31-39 (1989)). In yet another
embodiment, the polynucleotide sequences described herein can be
expressed in mammalian cells using a mammalian expression vector.
Other suitable expression systems for both prokaryotic and
eukaryotic cells are well known in the art; see, e.g., Sambrook et
al., "Molecular Cloning: A Laboratory Manual," second edition, Cold
Spring Harbor Laboratory, (1989).
As used herein "Acyl-CoA" refers to an acyl thioester formed
between the carbonyl carbon of alkyl chain and the sulfhydryl group
of the 4'-phosphopantethionyl moiety of coenzyme A (CoA), which has
the formula R--C(O)S-CoA, where R is any alkyl group having at
least 4 carbon atoms.
As used herein "acyl-ACP" refers to an acyl thioester formed
between the carbonyl carbon of alkyl chain and the sulfhydryl group
of the phosphopantetheinyl moiety of an acyl carrier protein (ACP).
The phosphopantetheinyl moiety is post-translationally attached to
a conserved serine residue on the ACP by the action of holo-acyl
carrier protein synthase (ACPS), a phosphopantetheinyl transferase.
In some embodiments an acyl-ACP is an intermediate in the synthesis
of fully saturated acyl-ACPs. In other embodiments an acyl-ACP is
an intermediate in the synthesis of unsaturated acyl-ACPs. In some
embodiments, the carbon chain will have about 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26
carbons. Each of these acyl-ACPs are substrates for enzymes that
convert them to fatty acid derivatives.
As used herein, the term "fatty acid or derivative thereof" means a
"fatty acid" or a "fatty acid derivative." The term "fatty acid"
means a carboxylic acid having the formula RCOOH. R represents an
aliphatic group, preferably an alkyl group. R can comprise between
about 4 and about 22 carbon atoms. Fatty acids can be saturated,
monounsaturated, or polyunsaturated. In a preferred embodiment, the
fatty acid is made from a fatty acid biosynthetic pathway. The term
"fatty acid derivative" means products made in part from the fatty
acid biosynthetic pathway of the production host organism. "Fatty
acid derivative" also includes products made in part from acyl-ACP
or acyl-ACP derivatives. Exemplary fatty acid derivatives include,
for example, acyl-CoA, fatty aldehydes, short and long chain
alcohols, hydrocarbons, and esters (e.g., waxes, fatty acid esters,
or fatty esters).
As used herein, the term "fatty acid biosynthetic pathway" means a
biosynthetic pathway that produces fatty acid derivatives, for
example, fatty alcohols. The fatty acid biosynthetic pathway
includes fatty acid synthases that can be engineered to produce
fatty acids, and in some embodiments can be expressed with
additional enzymes to produce fatty acid derivatives, such as fatty
alcohols having desired characteristics.
As used herein, "fatty aldehyde" means an aldehyde having the
formula RCHO characterized by a carbonyl group (C.dbd.O). In some
embodiments, the fatty aldehyde is any aldehyde made from a fatty
alcohol. In certain embodiments, the R group is at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 13, at least 14, at least 15, at least
16, at least 17, at least 18, or at least 19, carbons in length.
Alternatively, or in addition, the R group is 20 or less, 19 or
less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less,
13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or
less, 7 or less, or 6 or less carbons in length. Thus, the R group
can have an R group bounded by any two of the above endpoints. For
example, the R group can be 6-16 carbons in length, 10-14 carbons
in length, or 12-18 carbons in length. In some embodiments, the
fatty aldehyde is a C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10,
C.sub.11, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16,
C.sub.17, C.sub.18, C.sub.19, C.sub.20, C.sub.21, C.sub.22,
C.sub.23, C.sub.24, C.sub.25, or a C.sub.26 fatty aldehyde. In
certain embodiments, the fatty aldehyde is a C.sub.6, C.sub.8,
C.sub.10, C.sub.12, C.sub.13, C.sub.14, C.sub.15, C.sub.16,
C.sub.17, or C.sub.18 fatty aldehyde.
As used herein, "fatty alcohol" means an alcohol having the formula
ROH. In some embodiments, the R group is at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 11, at least
12, at least 13, at least 14, at least 15, at least 16, at least
17, at least 18, or at least 19, carbons in length. Alternatively,
or in addition, the R group is 20 or less, 19 or less, 18 or less,
17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or
less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, or 6
or less carbons in length. Thus, the R group can have an R group
bounded by any two of the above endpoints. For example, the R group
can be 6-16 carbons in length, 10-14 carbons in length, or 12-18
carbons in length. In some embodiments, the fatty alcohol is a
C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12,
C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18,
C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24,
C.sub.25, or a C.sub.26 fatty alcohol. In certain embodiments, the
fatty alcohol is a C.sub.6, C.sub.8, C.sub.10, C.sub.12, C.sub.13,
C.sub.14, C.sub.15, C.sub.16, C.sub.17, or C.sub.18 fatty
alcohol.
A "fatty alcohol composition" as referred to herein is produced by
a recombinant host cell and typically comprises a mixture of fatty
alcohols. In some cases, the mixture includes more than one type of
product (e.g., fatty alcohols and fatty acids). In other cases, the
fatty acid derivative compositions may comprise, for example, a
mixture of fatty alcohols with various chain lengths and saturation
or branching characteristics. In still other cases, the fatty
alcohol composition comprises a mixture of both more than one type
of product and products with various chain lengths and saturation
or branching characteristics.
A host cell engineered to produce a fatty aldehyde will typically
convert some of the fatty aldehyde to a fatty alcohol. When a host
cell, which produces fatty alcohols is engineered to express a
polynucleotide encoding an ester synthase, wax esters are produced.
In one embodiment, fatty alcohols are made from a fatty acid
biosynthetic pathway. As an example, Acyl-ACP can be converted to
fatty acids via the action of a thioesterase (e.g., E. coli TesA),
which are converted to fatty aldehydes and fatty alcohols via the
action of a carboxylic acid reductase (e.g., E. coli CarB).
Conversion of fatty aldehydes to fatty alcohols can be further
facilitated, for example, via the action of a fatty alcohol
biosynthetic polypeptide. In some embodiments, a gene encoding a
fatty alcohol biosynthetic polypeptide is expressed or
overexpressed in the host cell. In certain embodiments, the fatty
alcohol biosynthetic polypeptide has aldehyde reductase or alcohol
dehydrogenase activity. Examples of alcohol dehydrogenase
polypeptides useful in accordance with the disclosure include, but
are not limited to AlrA of Acinetobacter sp. M-1 (SEQ ID NO: 3) or
AlrA homologs, such as AlrAadp1 (SEQ ID NO:4) and endogenous E.
coli alcohol dehydrogenases such as YjgB, (AAC77226) (SEQ ID NO:
5), DkgA (NP_417485), DkgB (NP_414743), YdjL (AAC74846), YdjJ
(NP_416288), AdhP (NP_415995), YhdH (NP_417719), YahK (NP_414859),
YphC (AAC75598), YqhD (446856) and YbbO [AAC73595.1]. Additional
examples are described in International Patent Application
Publication Nos. WO2007/136762, WO2008/119082 and WO2010/062480,
each of which is expressly incorporated by reference herein. In
certain embodiments, the fatty alcohol biosynthetic polypeptide has
aldehyde reductase or alcohol dehydrogenase activity (EC
1.1.1.1).
As used herein, the term "alcohol dehydrogenase" refers to a
polypeptide capable of catalyzing the conversion of a fatty
aldehyde to an alcohol (e.g., fatty alcohol). One of ordinary skill
in the art will appreciate that certain alcohol dehydrogenases are
capable of catalyzing other reactions as well, and these
non-specific alcohol dehydrogenases also are encompassed by the
term "alcohol dehydrogenase." The R group of a fatty acid, fatty
aldehyde, or fatty alcohol can be a straight chain or a branched
chain. Branched chains may have more than one point of branching
and may include cyclic branches. In some embodiments, the branched
fatty acid, branched fatty aldehyde, or branched fatty alcohol is a
C.sub.6, C.sub.7, C.sub.8, C.sub.9, C.sub.10, C.sub.11, C.sub.12,
C.sub.13, C.sub.14, C.sub.15, C.sub.16, C.sub.17, C.sub.18,
C.sub.19, C.sub.20, C.sub.21, C.sub.22, C.sub.23, C.sub.24,
C.sub.25, or a C.sub.26 branched fatty acid, branched fatty
aldehyde, or branched fatty alcohol. In particular embodiments, the
branched fatty acid, branched fatty aldehyde, or branched fatty
alcohol is a C.sub.6, C.sub.8, C.sub.10, C.sub.12, C.sub.13,
C.sub.14, C.sub.15, C.sub.16, C.sub.17, or Cis branched fatty acid,
branched fatty aldehyde, or branched fatty alcohol. In certain
embodiments, the hydroxyl group of the branched fatty acid,
branched fatty aldehyde, or branched fatty alcohol is in the
primary (CO position. In certain embodiments, the branched fatty
acid, branched fatty aldehyde, or branched fatty alcohol is an
iso-fatty acid, iso-fatty aldehyde, or iso-fatty alcohol, or an
antesio-fatty acid, an anteiso-fatty aldehyde, or anteiso-fatty
alcohol. In exemplary embodiments, the branched fatty acid,
branched fatty aldehyde, or branched fatty alcohol is selected from
iso-C.sub.7:0, iso-C.sub.8:0, iso-C.sub.9:0, iso-C.sub.10:0,
iso-C.sub.11:0, iso-C.sub.12:0, iso-C.sub.13:0, iso-C.sub.14:0,
iso-C.sub.15:0, iso-C.sub.16:0, iso-C.sub.17:0, iso-C.sub.18:0,
iso-C.sub.19:0, anteiso-C.sub.70, anteiso-C.sub.8:0,
anteiso-C.sub.9:0, anteiso-C.sub.10:0, anteiso-C.sub.11:0,
anteiso-C.sub.12:0, anteiso-C.sub.13:0, anteiso-C.sub.14:0,
anteiso-C.sub.15:0, anteiso-C.sub.16:0, anteiso-C.sub.17:0,
anteiso-C.sub.18:0, and anteiso-C.sub.19:0 branched fatty acid,
branched fatty aldehyde or branched fatty alcohol. The R group of a
branched or unbranched fatty acid, branched or unbranched fatty
aldehyde, or branched or unbranched fatty alcohol can be saturated
or unsaturated. If unsaturated, the R group can have one or more
than one point of unsaturation. In some embodiments, the
unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated
fatty alcohol is a monounsaturated fatty acid, monounsaturated
fatty aldehyde, or monounsaturated fatty alcohol. In certain
embodiments, the unsaturated fatty acid, unsaturated fatty
aldehyde, or unsaturated fatty alcohol is a C6:1, C7:1, C8:1, C9:1,
C10:1, C11:1, C12:1, C13:1, C14:1, C15:1, C16:1, C17:1, C18:1,
C19:1, C20:1, C21:1, C22:1, C23:1, C24:1, C25:1, or a C26:1
unsaturated fatty acid, unsaturated fatty aldehyde, or unsaturated
fatty alcohol. In certain preferred embodiments, the unsaturated
fatty acid, unsaturated fatty aldehyde, or unsaturated fatty
alcohol is C10:1, C12:1, C14:1, C16:1, or C18:1. In yet other
embodiments, the unsaturated fatty acid, unsaturated fatty
aldehyde, or unsaturated fatty alcohol is unsaturated at the
omega-7 position. In certain embodiments, the unsaturated fatty
acid, unsaturated fatty aldehyde, or unsaturated fatty alcohol
comprises a cis double bond.
As used herein, a recombinant or engineered "host cell" is a host
cell, e.g., a microorganism that has been modified such that it
produces fatty alcohols. In some embodiments, the recombinant host
cell comprises one or more polynucleotides, each polynucleotide
encoding a polypeptide having fatty aldehyde and/or fatty alcohol
biosynthetic enzyme activity, wherein the recombinant host cell
produces a fatty alcohol composition when cultured in the presence
of a carbon source under conditions effective to express the
polynucleotides.
As used herein, the term "clone" typically refers to a cell or
group of cells descended from and essentially genetically identical
to a single common ancestor, for example, the bacteria of a cloned
bacterial colony arose from a single bacterial cell.
As used herein, the term "culture" typical refers to a liquid media
comprising viable cells. In one embodiment, a culture comprises
cells reproducing in a predetermined culture media under controlled
conditions, for example, a culture of recombinant host cells grown
in liquid media comprising a selected carbon source and nitrogen.
"Culturing" or "cultivation" refers to growing a population of
microbial cells under suitable conditions in a liquid or solid
medium. In particular embodiments, culturing refers to the
fermentative bioconversion of a substrate to an end-product.
Culturing media are well known and individual components of such
culture media are available from commercial sources, e.g., under
the Difco.TM. and BBL.TM. trademarks. In one non-limiting example,
the aqueous nutrient medium is a "rich medium" comprising complex
sources of nitrogen, salts, and carbon, such as YP medium,
comprising 10 g/L of peptone and 10 g/L yeast extract of such a
medium. The host cell can be additionally engineered to assimilate
carbon efficiently and use cellulosic materials as carbon sources
according to methods described for example in U.S. Pat. Nos.
5,000,000; 5,028,539; 5,424,202; 5,482,846; 5,602,030 and
WO2010127318, each of which is expressly incorporated by reference
herein. In addition, the host cell can be engineered to express an
invertase so that sucrose can be used as a carbon source.
As used herein, the term "under conditions effective to express
said heterologous nucleotide sequences" means any conditions that
allow a host cell to produce a desired fatty aldehyde or fatty
alcohol. Suitable conditions include, for example, fermentation
conditions.
As used herein, "modified" or an "altered level of" activity of a
protein, for example an enzyme, in a recombinant host cell refers
to a difference in one or more characteristics in the activity
determined relative to the parent or native host cell. Typically
differences in activity are determined between a recombinant host
cell, having modified activity, and the corresponding wild-type
host cell (e.g., comparison of a culture of a recombinant host cell
relative to wild-type host cell). Modified activities can be the
result of, for example, modified amounts of protein expressed by a
recombinant host cell (e.g., as the result of increased or
decreased number of copies of DNA sequences encoding the protein,
increased or decreased number of mRNA transcripts encoding the
protein, and/or increased or decreased amounts of protein
translation of the protein from mRNA); changes in the structure of
the protein (e.g., changes to the primary structure, such as,
changes to the protein's coding sequence that result in changes in
substrate specificity, changes in observed kinetic parameters); and
changes in protein stability (e.g., increased or decreased
degradation of the protein). In some embodiments, the polypeptide
is a mutant or a variant of any of the polypeptides described
herein. In certain instances, the coding sequences for the
polypeptides described herein are codon optimized for expression in
a particular host cell. For example, for expression in E. coli, one
or more codons can be optimized as described in, e.g., Grosjean et
al., Gene 18:199-209 (1982).
The term "regulatory sequences" as used herein typically refers to
a sequence of bases in DNA, operably-linked to DNA sequences
encoding a protein that ultimately controls the expression of the
protein. Examples of regulatory sequences include, but are not
limited to, RNA promoter sequences, transcription factor binding
sequences, transcription termination sequences, modulators of
transcription (such as enhancer elements), nucleotide sequences
that affect RNA stability, and translational regulatory sequences
(such as, ribosome binding sites (e.g., Shine-Dalgarno sequences in
prokaryotes or Kozak sequences in eukaryotes), initiation codons,
termination codons).
As used herein, the phrase "the expression of said nucleotide
sequence is modified relative to the wild type nucleotide
sequence," means an increase or decrease in the level of expression
and/or activity of an endogenous nucleotide sequence or the
expression and/or activity of a heterologous or non-native
polypeptide-encoding nucleotide sequence. As used herein, the term
"overexpress" means to express or cause to be expressed a
polynucleotide or polypeptide in a cell at a greater concentration
than is normally expressed in a corresponding wild-type cell under
the same conditions.
The terms "altered level of expression" and "modified level of
expression" are used interchangeably and mean that a
polynucleotide, polypeptide, or hydrocarbon is present in a
different concentration in an engineered host cell as compared to
its concentration in a corresponding wild-type cell under the same
conditions.
As used herein, the term "titer" refers to the quantity of fatty
aldehyde or fatty alcohol produced per unit volume of host cell
culture. In any aspect of the compositions and methods described
herein, a fatty alcohol is produced at a titer of about 25 mg/L,
about 50 mg/L, about 75 mg/L, about 100 mg/L, about 125 mg/L, about
150 mg/L, about 175 mg/L, about 200 mg/L, about 225 mg/L, about 250
mg/L, about 275 mg/L, about 300 mg/L, about 325 mg/L, about 350
mg/L, about 375 mg/L, about 400 mg/L, about 425 mg/L, about 450
mg/L, about 475 mg/L, about 500 mg/L, about 525 mg/L, about 550
mg/L, about 575 mg/L, about 600 mg/L, about 625 mg/L, about 650
mg/L, about 675 mg/L, about 700 mg/L, about 725 mg/L, about 750
mg/L, about 775 mg/L, about 800 mg/L, about 825 mg/L, about 850
mg/L, about 875 mg/L, about 900 mg/L, about 925 mg/L, about 950
mg/L, about 975 mg/L, about 1000 mg/L, about 1050 mg/L, about 1075
mg/L, about 1100 mg/L, about 1125 mg/L, about 1150 mg/L, about 1175
mg/L, about 1200 mg/L, about 1225 mg/L, about 1250 mg/L, about 1275
mg/L, about 1300 mg/L, about 1325 mg/L, about 1350 mg/L, about 1375
mg/L, about 1400 mg/L, about 1425 mg/L, about 1450 mg/L, about 1475
mg/L, about 1500 mg/L, about 1525 mg/L, about 1550 mg/L, about 1575
mg/L, about 1600 mg/L, about 1625 mg/L, about 1650 mg/L, about 1675
mg/L, about 1700 mg/L, about 1725 mg/L, about 1750 mg/L, about 1775
mg/L, about 1800 mg/L, about 1825 mg/L, about 1850 mg/L, about 1875
mg/L, about 1900 mg/L, about 1925 mg/L, about 1950 mg/L, about 1975
mg/L, about 2000 mg/L (2 g/L), 3 g/L, 5 g/L, 10 g/L, 20 g/L, 30
g/L, 40 g/L, 50 g/L, 60 g/L, 70 g/L, 80 g/L, 90 g/L, 100 g/L or a
range bounded by any two of the foregoing values. In other
embodiments, a fatty aldehyde or fatty alcohol is produced at a
titer of more than 100 g/L, more than 200 g/L, more than 300 g/L,
or higher, such as 500 g/L, 700 g/L, 1000 g/L, 1200 g/L, 1500 g/L,
or 2000 g/L. The preferred titer of fatty aldehyde or fatty alcohol
produced by a recombinant host cell according to the methods of the
disclosure is from 5 g/L to 200 g/L, 10 g/L to 150 g/L, 20 g/L to
120 g/L and 30 g/L to 100 g/L.
As used herein, the term "yield of the fatty aldehyde or fatty
alcohol produced by a host cell" refers to the efficiency by which
an input carbon source is converted to product (i.e., fatty alcohol
or fatty aldehyde) in a host cell. Host cells engineered to produce
fatty alcohols and/or fatty aldehydes according to the methods of
the disclosure have a yield of at least 3%, at least 4%, at least
5%, at least 6%, at least 7%, at least 8%, at least 9%, at least
10%, at least 11%, at least 12%, at least 13%, at least 14%, at
least 15%, at least 16%, at least 17%, at least 18%, at least 19%,
at least 20%, at least 21%, at least 22%, at least 23%, at least
24%, at least 25%, at least 26%, at least 27%, at least 28%, at
least 29%, or at least 30% or a range bounded by any two of the
foregoing values. In other embodiments, a fatty aldehyde or fatty
alcohol is produced at a yield of more than 30%, 40%, 50%, 60%,
70%, 80%, 90% or more. Alternatively, or in addition, the yield is
about 30% or less, about 27% or less, about 25% or less, or about
22% or less. Thus, the yield can be bounded by any two of the above
endpoints. For example, the yield of the fatty alcohol or fatty
aldehyde produced by the recombinant host cell according to the
methods of the disclosure can be 5% to 15%, 10% to 25%, 10% to 22%,
15% to 27%, 18% to 22%, 20% to 28%, or 20% to 30%. The preferred
yield of fatty alcohol produced by the recombinant host cell
according to the methods of the disclosure is from 10% to 30%.
As used herein, the term "productivity" refers to the quantity of
fatty aldehyde or fatty alcohol produced per unit volume of host
cell culture per unit time. In any aspect of the compositions and
methods described herein, the productivity of fatty aldehyde or
fatty alcohol produced by a recombinant host cell is at least 100
mg/L/hour, at least 200 mg/L/hour.sub.0, at least 300 mg/L/hour, at
least 400 mg/L/hour, at least 500 mg/L/hour, at least 600
mg/L/hour, at least 700 mg/L/hour, at least 800 mg/L/hour, at least
900 mg/L/hour, at least 1000 mg/L/hour, at least 1100 mg/L/hour, at
least 1200 mg/L/hour, at least 1300 mg/L/hour, at least 1400
mg/L/hour, at least 1500 mg/L/hour, at least 1600 mg/L/hour, at
least 1700 mg/L/hour, at least 1800 mg/L/hour, at least 1900
mg/L/hour, at least 2000 mg/L/hour, at least 2100 mg/L/hour, at
least 2200 mg/L/hour, at least 2300 mg/L/hour, at least 2400
mg/L/hour, or at least 2500 mg/L/hour. Alternatively, or in
addition, the productivity is 2500 mg/L/hour or less, 2000
mg/L/OD.sub.600 or less, 1500 mg/L/OD.sub.600 or less, 120
mg/L/hour, or less, 1000 mg/L/hour or less, 800 mg/L/hour, or less,
or 600 mg/L/hour or less. Thus, the productivity can be bounded by
any two of the above endpoints. For example, the productivity can
be 3 to 30 mg/L/hour.sub.0, 6 to 20 mg/L/hour, or 15 to 30
mg/L/hour. The preferred productivity of a fatty aldehyde or fatty
alcohol produced by a recombinant host cell according to the
methods of the disclosure is selected from 500 mg/L/hour to 2500
mg/L/hour, or from 700 mg/L/hour to 2000 mg/L/hour.
The terms "total fatty species" and "total fatty acid product" may
be used interchangeably herein with reference to the total amount
of fatty alcohols, fatty aldehydes, free fatty acids, and fatty
esters present in a sample as evaluated by GC-FID as described in
International Patent Application Publication WO 2008/119082.
Samples may contain one, two, three, or four of these compounds
depending on the context.
As used herein, the term "glucose utilization rate" means the
amount of glucose used by the culture per unit time, reported as
grams/liter/hour (g/L/hr).
As used herein, the term "carbon source" refers to a substrate or
compound suitable to be used as a source of carbon for prokaryotic
or simple eukaryotic cell growth. Carbon sources can be in various
forms, including, but not limited to polymers, carbohydrates,
acids, alcohols, aldehydes, ketones, amino acids, peptides, and
gases (e.g., CO and CO.sub.2). Exemplary carbon sources include,
but are not limited to, monosaccharides, such as glucose, fructose,
mannose, galactose, xylose, and arabinose; oligosaccharides, such
as fructo-oligosaccharide and galacto-oligosaccharide;
polysaccharides such as starch, cellulose, pectin, and xylan;
disaccharides, such as sucrose, maltose, cellobiose, and turanose;
cellulosic material and variants such as hemicelluloses, methyl
cellulose and sodium carboxymethyl cellulose; saturated or
unsaturated fatty acids, succinate, lactate, and acetate; alcohols,
such as ethanol, methanol, and glycerol, or mixtures thereof. The
carbon source can also be a product of photosynthesis, such as
glucose. In certain preferred embodiments, the carbon source is
biomass. In other preferred embodiments, the carbon source is
glucose. In other preferred embodiments the carbon source is
sucrose.
As used herein, the term "biomass" refers to any biological
material from which a carbon source is derived. In some
embodiments, a biomass is processed into a carbon source, which is
suitable for bioconversion. In other embodiments, the biomass does
not require further processing into a carbon source. The carbon
source can be converted into a biofuel. An exemplary source of
biomass is plant matter or vegetation, such as corn, sugar cane, or
switchgrass. Another exemplary source of biomass is metabolic waste
products, such as animal matter (e.g., cow manure). Further
exemplary sources of biomass include algae and other marine plants.
Biomass also includes waste products from industry, agriculture,
forestry, and households, including, but not limited to,
fermentation waste, ensilage, straw, lumber, sewage, garbage,
cellulosic urban waste, and food leftovers. The term "biomass" also
can refer to sources of carbon, such as carbohydrates (e.g.,
monosaccharides, disaccharides, or polysaccharides).
As used herein, the term "isolated," with respect to products (such
as fatty acids and derivatives thereof) refers to products that are
separated from cellular components, cell culture media, or chemical
or synthetic precursors. The fatty acids and derivatives thereof
produced by the methods described herein can be relatively
immiscible in the fermentation broth, as well as in the cytoplasm.
Therefore, the fatty acids and derivatives thereof can collect in
an organic phase either intracellularly or extracellularly.
As used herein, the terms "purify," "purified," or "purification"
mean the removal or isolation of a molecule from its environment
by, for example, isolation or separation. "Substantially purified"
molecules are at least about 60% free (e.g., at least about 70%
free, at least about 75% free, at least about 85% free, at least
about 90% free, at least about 95% free, at least about 97% free,
at least about 99% free) from other components with which they are
associated. As used herein, these terms also refer to the removal
of contaminants from a sample. For example, the removal of
contaminants can result in an increase in the percentage of a fatty
aldehyde or a fatty alcohol in a sample. For example, when a fatty
aldehyde or a fatty alcohol is produced in a recombinant host cell,
the fatty aldehyde or fatty alcohol can be purified by the removal
of recombinant host cell proteins. After purification, the
percentage of a fatty aldehyde or a fatty alcohol in the sample is
increased. The terms "purify," "purified," and "purification" are
relative terms which do not require absolute purity. Thus, for
example, when a fatty aldehyde or a fatty alcohol is produced in
recombinant host cells, a purified fatty aldehyde or a purified
fatty alcohol is a fatty aldehyde or a fatty alcohol that is
substantially separated from other cellular components (e.g.,
nucleic acids, polypeptides, lipids, carbohydrates, or other
hydrocarbons).
Strain Improvements
In order to meet very high targets for titer, yield, and/or
productivity of fatty alcohols, a number of modifications were made
to the production host cells. FadR is a key regulatory factor
involved in fatty acid degradation and fatty acid biosynthesis
pathways (Cronan et al., Mol. Microbiol., 29(4): 937-943 (1998)).
The E. coli ACS enzyme FadD and the fatty acid transport protein
FadL are essential components of a fatty acid uptake system. FadL
mediates transport of fatty acids into the bacterial cell, and FadD
mediates formation of acyl-CoA esters. When no other carbon source
is available, exogenous fatty acids are taken up by bacteria and
converted to acyl-CoA esters, which can bind to the transcription
factor FadR and derepress the expression of the fad genes that
encode proteins responsible for fatty acid transport (FadL),
activation (FadD), and .beta.-oxidation (FadA, FadB, FadE, and
FadH). When alternative sources of carbon are available, bacteria
synthesize fatty acids as acyl-ACPs, which are used for
phospholipid synthesis, but are not substrates for
.beta.-oxidation. Thus, acyl-CoA and acyl-ACP are both independent
sources of fatty acids that can result in different end-products
(Caviglia et al., J. Biol. Chem., 279(12): 1163-1169 (2004)). U.S.
Provisional Application No. 61/470,989 describes improved methods
of producing fatty acid derivatives in a host cell which is
genetically engineered to have an altered level of expression of a
FadR polypeptide as compared to the level of expression of the FadR
polypeptide in a corresponding wild-type host cell.
There are conflicting speculations in the art as to the limiting
factors of fatty acid biosynthesis in host cells, such as E. coli.
One approach to increasing the flux through fatty acid biosynthesis
is to manipulate various enzymes in the pathway (FIGS. 1 and 2).
The supply of acyl-ACPs from acetyl-CoA via the acetyl-CoA
carboxylase (acc) complex (FIG. 3) and fatty acid biosynthetic
(fab) pathway may limit the rate of fatty alcohol production. In
one exemplary approach detailed in Example 2, the effect of
overexpression of Corynebacterium glutamicum accABCD (.+-.birA)
demonstrated that such genetic modifications can lead to increased
acetyl-coA and malonyl-CoA in E. coli. One possible reason for a
low rate of flux through fatty acid biosynthesis is a limited
supply of precursors, namely acetyl-CoA and, in particular,
malonyl-CoA, and the main precursors for fatty acid biosynthesis.
Example 3 describes the construction of fab operons that encode
enzymes in the biosynthetic pathway for conversion of malonyl-CoA
into acyl-ACPs and integration into the chromosome of an E. coli
host cell. In yet another approach detailed in Example 4, mutations
in the rph and ilvG genes in the E. coli host cell were shown to
result in higher free fatty acid (FFA) production, which translated
into higher production of fatty alcohol. In still another approach,
transposon mutagenesis and high-throughput screening was done to
find beneficial mutations that increase the titer or yield. Example
5 describes how a transposon insertion in the yijP gene can improve
the fatty alcohol yield in shake flask and fed-batch
fermentations.
Carboxylic Acid Reductase (CAR)
Recombinant host cells have been engineered to produce fatty
alcohols by expressing a thioesterase, which catalyzes the
conversion of acyl-ACPs into free fatty acids (FFAs) and a
carboxylic acid reductase (CAR), which converts free fatty acids
into fatty aldehydes. Native (endogenous) aldehyde reductases
present in the host cell (e.g., E. coli) can convert fatty
aldehydes into fatty alcohols. Exemplary thioesterases are
described for example in US Patent Publication No. 20100154293,
expressly incorporated by reference herein. CarB, is an exemplary
carboxylic acid reductase, a key enzyme in the fatty alcohol
production pathway. WO2010/062480 describes a BLAST search using
the NRRL 5646 CAR amino acid sequence (Genpept accession AAR91681)
(SEQ ID NO: 6) as the query sequence, and use thereof in
identification of approximately 20 homologous sequences.
The terms "carboxylic acid reductase," "CAR," and "fatty aldehyde
biosynthetic polypeptide" are used interchangeably herein. In
practicing the disclosure, a gene encoding a carboxylic acid
reductase polypeptide is expressed or overexpressed in the host
cell. In some embodiments, the CarB polypeptide has the amino acid
sequence of SEQ ID NO: 7. In other embodiments, the CarB
polypeptide is a variant or mutant of SEQ ID NO: 7. In certain
embodiments, the CarB polypeptide is from a mammalian cell, plant
cell, insect cell, yeast cell, fungus cell, filamentous fungi cell,
a bacterial cell, or any other organism. In some embodiments, the
bacterial cell is a mycobacterium selected from the group
consisting of Mycobacterium smegmatis, Mycobacterium abscessus,
Mycobacterium avium, Mycobacterium bovis, Mycobacterium
tuberculosis, Mycobacterium leprae, Mycobacterium marinum, and
Mycobacterium ulcerans. In other embodiments, the bacterial cell is
from a Nocardia species, for example, Nocardia NRRL 5646, Nocardia
farcinica, Streptomyces griseus, Salinispora arenicola, or
Clavibacter michiganenesis. In other embodiments, the CarB
polypeptide is a homologue of CarB having an amino acid sequence
that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical
to the amino acid sequence of SEQ ID NO: 7. The identity of a CarB
polypeptide having at least about 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or
99% identity to the amino acid sequence of SEQ ID NO: 7 is not
particularly limited, and one of ordinary skill in the art can
readily identify homologues of E. coli MG1655 derived-CarB and
determine its function using the methods described herein. In other
embodiments, the CarB polypeptide contains a mutation at amino acid
number 3, 12, 20, 28, 46, 74, 103, 191, 288, 473, 827, 926, 927,
930 or 1128 of SEQ ID NO: 7. Exemplary mutations are detailed in
Table 10. Preferred fragments or mutants of a polypeptide retain
some or all of the biological function (e.g., enzymatic activity)
of the corresponding wild-type polypeptide. In some embodiments,
the fragment or mutant retains at least about 75%, at least about
80%, at least about 90%, at least about 95%, or at least about 98%
or more of the biological function of the corresponding wild-type
polypeptide. In other embodiments, the fragment or mutant retains
about 100% of the biological function of the corresponding
wild-type polypeptide. Guidance in determining which amino acid
residues may be substituted, inserted, or deleted without affecting
biological activity may be found using computer programs well known
in the art, for example, LASERGENE.TM. software (DNASTAR, Inc.,
Madison, Wis.).
In yet other embodiments, a fragment or mutant exhibits increased
biological function as compared to a corresponding wild-type
polypeptide. For example, a fragment or mutant may display at least
about a 10%, at least about a 25%, at least about a 50%, at least
about a 75%, or at least about a 90% improvement in enzymatic
activity as compared to the corresponding wild-type polypeptide. In
other embodiments, the fragment or mutant displays at least about
100% (e.g., at least about 200%, or at least about 500%)
improvement in enzymatic activity as compared to the corresponding
wild-type polypeptide. It is understood that the polypeptides
described herein may have additional conservative or non-essential
amino acid substitutions, which do not have a substantial effect on
the polypeptide function. Whether or not a particular substitution
will be tolerated (i.e., will not adversely affect desired
biological function, such as DNA binding or enzyme activity) can be
determined as described in Bowie et al. (Science, 247: 1306-1310
(1990)).
As a result of the methods and variant enzymes of the present
disclosure, one or more of the titer, yield, and/or productivity of
the fatty acid or derivative thereof produced by the engineered
host cell having an altered level of expression of a CarB
polypeptide is increased relative to that of the corresponding
wild-type host cell. To allow for maximum conversion of C12 and C14
fatty acids into fatty alcohols, CarB must be expressed at
sufficient activity. An improved recombinant host cell would have a
CAR enzyme that is expressed from, for example, the E. coli
chromosome. As shown in Example 6, cells expressing the CarB enzyme
from the chromosome have more carboxylic acid reductase activity
relative to the original CarB and are able to convert more C12 and
C14 fatty acids into fatty alcohols. CarB is a large gene (3.5 kb)
and increases plasmid size considerably, making it difficult to use
a pCL plasmid to test new genes during strain development.
Approaches to increasing the activity of CarB, include increasing
its solubility, stability, expression and/or functionality. In one
exemplary approach, a fusion protein that contains 6 histidines and
a thrombin cleavage site at the N-terminus of CarB is produced.
This enzyme differs from CarB by an additional 60 nucleotides at
the N-terminus, and is named CarB60. When CarB or CarB60 are
expressed from the E. coli chromosome under control of the pTRC
promoter, cells containing CarB60 have increased total cellular
carboxylic acid reductase activity and convert more C12 and C14
free fatty acids (FFAs) into fatty alcohols. One of skill in the
art will appreciate that this is one example of molecular
engineering in order to achieve a greater conversion of C12 and C14
free fatty acids (FFAs) into fatty alcohols as illustrated in
Example 6 (supra). Similar approaches are encompassed herein (see
Example 7).
Phosphopantetheine transferases (PPTases) (EC 2.7.8.7) catalyze the
transfer of 4'-phosphopantetheine from CoA to a substrate. Nocardia
Car, CarB and several homologues thereof contain a putative
attachment site for 4'-phosphopantetheine (PPT) (He et al., Appl.
Environ. Microbiol., 70(3): 1874-1881 (2004)). In some embodiments
of the disclosure, a PPTase is expressed or overexpressed in an
engineered host cell. In certain embodiments, the PPTase is EntD
from E. coli MG1655 (SEQ ID NO:8). In some embodiments, a
thioesterase and a carboxylic acid reductase are expressed or
overexpressed in an engineered host cell. In certain embodiments,
the thioesterase is tesA and the carboxylic acid reductase is carB.
In other embodiments, a thioesterase, a carboxylic acid reductase
and an alcohol dehydrogenase are expressed or overexpressed in an
engineered host cell. In certain embodiments, the thioesterase is
tesA, the carboxylic acid reductase is carB and the alcohol
dehydrogenase is alrAadp1 (GenPept accession number CAG70248.1)
from Acinetobacter baylyi ADP1 (SEQ ID NO: 4). In still other
embodiments, a thioesterase, a carboxylic acid reductase, a PPTase,
and an alcohol dehydrogenase are expressed or overexpressed in the
engineered host cell. In certain embodiments, the thioesterase is
tesA, the carboxylic acid reductase is carB, the PPTase is entD,
and the alcohol dehydrogenase is alrAadp1. In still further
embodiments, a modified host cell which expresses one or more of a
thioesterase, a CAR, a PPTase, and an alcohol dehydrogenase also
has one or more strain improvements. Exemplary strain improvements
include, but are not limited to expression or overexpression of an
acetyl-CoA carboxylase polypeptide, overexpression of a FadR
polypeptide, expression or overexpression of a heterologous iFAB
operon, or transposon insertion in the yijP gene or another gene,
or similar approaches. The disclosure also provides a fatty alcohol
composition produced by any of the methods described herein. A
fatty alcohol composition produced by any of the methods described
herein can be used directly as a starting materials for production
of other chemical compounds (e.g., polymers, surfactants, plastics,
textiles, solvents, adhesives, etc.), or personal care additives.
These compounds can also be used as feedstock for subsequent
reactions, for example, hydrogenation, catalytic cracking (e.g.,
via hydrogenation, pyrolisis, or both) to make other products.
Mutants or Variants
In some embodiments, the polypeptide expressed in a recombinant
host cell is a mutant or a variant of any of the polypeptides
described herein. The terms "mutant" and "variant" as used herein
refer to a polypeptide having an amino acid sequence that differs
from a wild-type polypeptide by at least one amino acid. For
example, the mutant can comprise one or more of the following
conservative amino acid substitutions: replacement of an aliphatic
amino acid, such as alanine, valine, leucine, and isoleucine, with
another aliphatic amino acid; replacement of a serine with a
threonine; replacement of a threonine with a serine; replacement of
an acidic residue, such as aspartic acid and glutamic acid, with
another acidic residue; replacement of a residue bearing an amide
group, such as asparagine and glutamine, with another residue
bearing an amide group; exchange of a basic residue, such as lysine
and arginine, with another basic residue; and replacement of an
aromatic residue, such as phenylalanine and tyrosine, with another
aromatic residue. In some embodiments, the mutant polypeptide has
about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70,
80, 90, 100, or more amino acid substitutions, additions,
insertions, or deletions. Preferred fragments or mutants of a
polypeptide retain some or all of the biological function (e.g.,
enzymatic activity) of the corresponding wild-type polypeptide. In
some embodiments, the fragment or mutant retains at least about
75%, at least about 80%, at least about 90%, at least about 95%, or
at least about 98% or more of the biological function of the
corresponding wild-type polypeptide. In other embodiments, the
fragment or mutant retains about 100% of the biological function of
the corresponding wild-type polypeptide. Guidance in determining
which amino acid residues may be substituted, inserted, or deleted
without affecting biological activity may be found using computer
programs well known in the art, for example, LASERGENE.TM. software
(DNASTAR, Inc., Madison, Wis.).
In yet other embodiments, a fragment or mutant exhibits increased
biological function as compared to a corresponding wild-type
polypeptide. For example, a fragment or mutant may display at least
a 10%, at least a 25%, at least a 50%, at least a 75%, or at least
a 90% improvement in enzymatic activity as compared to the
corresponding wild-type polypeptide. In other embodiments, the
fragment or mutant displays at least 100% (e.g., at least 200%, or
at least 500%) improvement in enzymatic activity as compared to the
corresponding wild-type polypeptide. It is understood that the
polypeptides described herein may have additional conservative or
non-essential amino acid substitutions, which do not have a
substantial effect on the polypeptide function. Whether or not a
particular substitution will be tolerated (i.e., will not adversely
affect desired biological function, such as carboxylic acid
reductase activity) can be determined as described in Bowie et al.
(Science, 247: 1306-1310 (1990)). A conservative amino acid
substitution is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine),
and aromatic side chains (e.g., tyrosine, phenylalanine,
tryptophan, histidine). Variants can be naturally occurring or
created in vitro. In particular, such variants can be created using
genetic engineering techniques, such as site directed mutagenesis,
random chemical mutagenesis, Exonuclease III deletion procedures,
or standard cloning techniques. Alternatively, such variants,
fragments, analogs, or derivatives can be created using chemical
synthesis or modification procedures.
Methods of making variants are well known in the art. These include
procedures in which nucleic acid sequences obtained from natural
isolates are modified to generate nucleic acids that encode
polypeptides having characteristics that enhance their value in
industrial or laboratory applications. In such procedures, a large
number of variant sequences having one or more nucleotide
differences with respect to the sequence obtained from the natural
isolate are generated and characterized. Typically, these
nucleotide differences result in amino acid changes with respect to
the polypeptides encoded by the nucleic acids from the natural
isolates. For example, variants can be prepared by using random and
site-directed mutagenesis. Random and site-directed mutagenesis are
described in, for example, Arnold, Curr. Opin. Biotech., 4: 450-455
(1993). Random mutagenesis can be achieved using error prone PCR
(see, e.g., Leung et al., Technique, 1: 11-15 (1989); and Caldwell
et al., PCR Methods Applic., 2: 28-33 (1992)). In error prone PCR,
PCR is performed under conditions where the copying fidelity of the
DNA polymerase is low, such that a high rate of point mutations is
obtained along the entire length of the PCR product. Briefly, in
such procedures, nucleic acids to be mutagenized (e.g., a
polynucleotide sequence encoding a carboxylic reductase enzyme) are
mixed with PCR primers, reaction buffer, MgCl.sub.2, MnCl.sub.2,
Taq polymerase, and an appropriate concentration of dNTPs for
achieving a high rate of point mutation along the entire length of
the PCR product. For example, the reaction can be performed using
20 fmoles of nucleic acid to be mutagenized, 30 pmole of each PCR
primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH
8.3), 0.01% gelatin, 7 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 5 units of
Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP.
PCR can be performed for 30 cycles of 94.degree. C. for 1 min,
45.degree. C. for 1 min, and 72.degree. C. for 1 min. However, it
will be appreciated that these parameters can be varied as
appropriate. The mutagenized nucleic acids are then cloned into an
appropriate vector, and the activities of the polypeptides encoded
by the mutagenized nucleic acids are evaluated (see Example 7).
Site-directed mutagenesis can be achieved using
oligonucleotide-directed mutagenesis to generate site-specific
mutations in any cloned DNA of interest. Oligonucleotide
mutagenesis is described in, for example, Reidhaar-Olson et al.,
Science, 241: 53-57 (1988). Briefly, in such procedures a plurality
of double stranded oligonucleotides bearing one or more mutations
to be introduced into the cloned DNA are synthesized and inserted
into the cloned DNA to be mutagenized (e.g., a polynucleotide
sequence encoding a CAR polypeptide). Clones containing the
mutagenized DNA are recovered, and the activities of the
polypeptides they encode are assessed. Another method for
generating variants is assembly PCR. Assembly PCR involves the
assembly of a PCR product from a mixture of small DNA fragments. A
large number of different PCR reactions occur in parallel in the
same vial, with the products of one reaction priming the products
of another reaction. Assembly PCR is described in, for example,
U.S. Pat. No. 5,965,408. Still another method of generating
variants is sexual PCR mutagenesis. In sexual PCR mutagenesis,
forced homologous recombination occurs between DNA molecules of
different, but highly related, DNA sequences in vitro as a result
of random fragmentation of the DNA molecule based on sequence
homology. This is followed by fixation of the crossover by primer
extension in a PCR reaction. Sexual PCR mutagenesis is described
in, for example, Stemmer, Proc. Natl. Acad. Sci., U.S.A., 91:
10747-10751 (1994).
Variants can also be created by in vivo mutagenesis. In some
embodiments, random mutations in a nucleic acid sequence are
generated by propagating the sequence in a bacterial strain, such
as an E. coli strain, which carries mutations in one or more of the
DNA repair pathways. Such "mutator" strains have a higher random
mutation rate than that of a wild-type strain. Propagating a DNA
sequence (e.g., a polynucleotide sequence encoding a CAR
polypeptide) in one of these strains will eventually generate
random mutations within the DNA. Mutator strains suitable for use
for in vivo mutagenesis are described in, for example,
International Patent Application Publication No. WO1991/016427.
Variants can also be generated using cassette mutagenesis. In
cassette mutagenesis, a small region of a double-stranded DNA
molecule is replaced with a synthetic oligonucleotide "cassette"
that differs from the native sequence. The oligonucleotide often
contains a completely and/or partially randomized native sequence.
Recursive ensemble mutagenesis can also be used to generate
variants. Recursive ensemble mutagenesis is an algorithm for
protein engineering (i.e., protein mutagenesis) developed to
produce diverse populations of phenotypically related mutants whose
members differ in amino acid sequence. This method uses a feedback
mechanism to control successive rounds of combinatorial cassette
mutagenesis. Recursive ensemble mutagenesis is described in, for
example, Arkin et al., Proc. Natl. Acad. Sci., U.S.A., 89:
7811-7815 (1992). In some embodiments, variants are created using
exponential ensemble mutagenesis. Exponential ensemble mutagenesis
is a process for generating combinatorial libraries with a high
percentage of unique and functional mutants, wherein small groups
of residues are randomized in parallel to identify, at each altered
position, amino acids which lead to functional proteins.
Exponential ensemble mutagenesis is described in, for example,
Delegrave et al., Biotech. Res, 11: 1548-1552 (1993). In some
embodiments, variants are created using shuffling procedures
wherein portions of a plurality of nucleic acids that encode
distinct polypeptides are fused together to create chimeric nucleic
acid sequences that encode chimeric polypeptides as described in,
for example, U.S. Pat. Nos. 5,965,408 and 5,939,250.
Insertional mutagenesis is mutagenesis of DNA by the insertion of
one or more bases. Insertional mutations can occur naturally,
mediated by virus or transposon, or can be artificially created for
research purposes in the lab, e.g., by transposon mutagenesis. When
exogenous DNA is integrated into that of the host, the severity of
any ensuing mutation depends entirely on the location within the
host's genome wherein the DNA is inserted. For example, significant
effects may be evident if a transposon inserts in the middle of an
essential gene, in a promoter region, or into a repressor or an
enhancer region. Transposon mutagenesis and high-throughput
screening was done to find beneficial mutations that increase the
titer or yield of fatty alcohol. The disclosure provides
recombinant host cells comprising (a) a polynucleotide sequence
encoding a carboxylic acid reductase comprising an amino acid
sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
sequence identity to the amino acid sequence of SEQ ID NO: 7 and
(b) a polynucleotide encoding a polypeptide having carboxylic acid
reductase activity, wherein the recombinant host cell is capable of
producing a fatty aldehyde or a fatty alcohol.
Engineering Host Cells
In some embodiments, a polynucleotide (or gene) sequence is
provided to a host cell by way of a recombinant vector, which
comprises a promoter operably linked to the polynucleotide
sequence. In certain embodiments, the promoter is a
developmentally-regulated, an organelle-specific, a
tissue-specific, an inducible, a constitutive, or a cell-specific
promoter. In some embodiments, the recombinant vector includes (a)
an expression control sequence operatively coupled to the
polynucleotide sequence; (b) a selection marker operatively coupled
to the polynucleotide sequence; (c) a marker sequence operatively
coupled to the polynucleotide sequence; (d) a purification moiety
operatively coupled to the polynucleotide sequence; (e) a secretion
sequence operatively coupled to the polynucleotide sequence; and
(f) a targeting sequence operatively coupled to the polynucleotide
sequence. The expression vectors described herein include a
polynucleotide sequence described herein in a form suitable for
expression of the polynucleotide sequence in a host cell. It will
be appreciated by those skilled in the art that the design of the
expression vector can depend on such factors as the choice of the
host cell to be transformed, the level of expression of polypeptide
desired, etc. The expression vectors described herein can be
introduced into host cells to produce polypeptides, including
fusion polypeptides, encoded by the polynucleotide sequences
described herein. Expression of genes encoding polypeptides in
prokaryotes, for example, E. coli, is most often carried out with
vectors containing constitutive or inducible promoters directing
the expression of either fusion or non-fusion polypeptides. Fusion
vectors add a number of amino acids to a polypeptide encoded
therein, usually to the amino- or carboxy-terminus of the
recombinant polypeptide. Such fusion vectors typically serve one or
more of the following three purposes: (1) to increase expression of
the recombinant polypeptide; (2) to increase the solubility of the
recombinant polypeptide; and (3) to aid in the purification of the
recombinant polypeptide by acting as a ligand in affinity
purification. Often, in fusion expression vectors, a proteolytic
cleavage site is introduced at the junction of the fusion moiety
and the recombinant polypeptide. This enables separation of the
recombinant polypeptide from the fusion moiety after purification
of the fusion polypeptide. Examples of such enzymes, and their
cognate recognition sequences, include Factor Xa, thrombin, and
enterokinase. Exemplary fusion expression vectors include pGEX
(Pharmacia Biotech, Inc., Piscataway, N.J.; Smith et al., Gene, 67:
31-40 (1988)), pMAL (New England Biolabs, Beverly, Mass.), and
pRITS (Pharmacia Biotech, Inc., Piscataway, N.J.), which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant polypeptide.
Examples of inducible, non-fusion E. coli expression vectors
include pTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d
(Studier et al., Gene Expression Technology: Methods in Enzymology
185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene
expression from the pTrc vector relies on host RNA polymerase
transcription from a hybrid trp-lac fusion promoter. Target gene
expression from the pET 11d vector relies on transcription from a
T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA
polymerase (T7 gni). This viral polymerase is supplied by host
strains BL21(DE3) or HMS174(DE3) from a resident .lamda. prophage
harboring a T7 gni gene under the transcriptional control of the
lacUV 5 promoter. Suitable expression systems for both prokaryotic
and eukaryotic cells are well known in the art; see, e.g., Sambrook
et al., "Molecular Cloning: A Laboratory Manual," second edition,
Cold Spring Harbor Laboratory, (1989). Examples of inducible,
non-fusion E. coli expression vectors include pTrc (Amann et al.,
Gene, 69: 301-315 (1988)) and PET 11d (Studier et al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif., pp. 60-89 (1990)). In certain embodiments, a
polynucleotide sequence of the disclosure is operably linked to a
promoter derived from bacteriophage T5. In one embodiment, the host
cell is a yeast cell. In this embodiment, the expression vector is
a yeast expression vector. Vectors can be introduced into
prokaryotic or eukaryotic cells via a variety of art-recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a
host cell. Suitable methods for transforming or transfecting host
cells can be found in, for example, Sambrook et al. (supra). For
stable transformation of bacterial cells, it is known that,
depending upon the expression vector and transformation technique
used, only a small fraction of cells will take-up and replicate the
expression vector. In some embodiments, in order to identify and
select these transformants, a gene that encodes a selectable marker
(e.g., resistance to an antibiotic) is introduced into the host
cells along with the gene of interest. Selectable markers include
those that confer resistance to drugs such as, but not limited to,
ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleic
acids encoding a selectable marker can be introduced into a host
cell on the same vector as that encoding a polypeptide described
herein or can be introduced on a separate vector. Cells stably
transformed with the introduced nucleic acid can be identified by
growth in the presence of an appropriate selection drug.
Production of Fatty Alcohol Compositions by Recombinant Host
Cells
Strategies to increase production of fatty alcohols by recombinant
host cells include increased flux through the fatty acid
biosynthetic pathway by overexpression of native fatty acid
biosynthesis genes and expression of exogenous fatty acid
biosynthesis genes from different organisms in an engineered
production host. Enhanced activity of relevant enzymes in the fatty
alcohol biosynthetic pathway, e.g., CAR, as well as other
strategies to optimize the growth and productivity of the host cell
may also be employed to maximize production. In some embodiments,
the recombinant host cell comprises a polynucleotide encoding a
polypeptide (an enzyme) having fatty alcohol biosynthetic activity
(i.e., a fatty alcohol biosynthetic polypeptide or a fatty alcohol
biosynthetic enzyme), and a fatty alcohol is produced by the
recombinant host cell. A composition comprising fatty alcohols (a
fatty alcohol composition) may be produced by culturing the
recombinant host cell in the presence of a carbon source under
conditions effective to express a fatty alcohol biosynthetic
enzyme. In some embodiments, the fatty alcohol composition
comprises fatty alcohols, however, a fatty alcohol composition may
comprise other fatty acid derivatives. Typically, the fatty alcohol
composition is recovered from the extracellular environment of the
recombinant host cell, i.e., the cell culture medium. In one
approach, recombinant host cells have been engineered to produce
fatty alcohols by expressing a thioesterase, which catalyzes the
conversion of acyl-ACPs into free fatty acids (FFAs) and a
carboxylic acid reductase (CAR), which converts free fatty acids
into fatty aldehydes. Native (endogenous) aldehyde reductases
present in the host cell (e.g., E. coli) can convert the fatty
aldehydes into fatty alcohols. In some embodiments, the fatty
alcohol is produced by expressing or overexpressing in the
recombinant host cell a polynucleotide encoding a polypeptide
having fatty alcohol biosynthetic activity which converts a fatty
aldehyde to a fatty alcohol. For example, an alcohol dehydrogenase
(also referred to herein as an aldehyde reductase, e.g., EC
1.1.1.1), may be used in practicing the disclosure. As used herein,
the term "alcohol dehydrogenase" refers to a polypeptide capable of
catalyzing the conversion of a fatty aldehyde to an alcohol (e.g.,
a fatty alcohol). One of ordinary skill in the art will appreciate
that certain alcohol dehydrogenases are capable of catalyzing other
reactions as well, and these non-specific alcohol dehydrogenases
also are encompassed by the term "alcohol dehydrogenase." Examples
of alcohol dehydrogenase polypeptides useful in accordance with the
disclosure include, but are not limited to AlrAadp1 (SEQ ID NO: 4)
or AlrA homologs and endogenous E. coli alcohol dehydrogenases such
as YjgB, (AAC77226) (SEQ ID NO: 5), DkgA (NP_417485), DkgB
(NP_414743), YdjL (AAC74846), YdjJ (NP_416288), AdhP (NP_415995),
YhdH (NP_417719), YahK (NP_414859), YphC (AAC75598), YqhD (446856)
and YbbO [AAC73595.1]. Additional examples are described in
International Patent Application Publication Nos. WO2007/136762,
WO2008/119082 and WO 2010/062480, each of which is expressly
incorporated by reference herein. In certain embodiments, the fatty
alcohol biosynthetic polypeptide has aldehyde reductase or alcohol
dehydrogenase activity (EC 1.1.1.1). In another approach,
recombinant host cells have been engineered to produce fatty
alcohols by expressing fatty alcohol forming acyl-CoA reductases or
fatty acyl reductases (FARs) which convert fatty acyl-thioester
substrates (e.g., fatty acyl-CoA or fatty acyl-ACP) to fatty
alcohols. In some embodiments, the fatty alcohol is produced by
expressing or overexpressing a polynucleotide encoding a
polypeptide having fatty alcohol forming acyl-CoA reductase (FAR)
activity in a recombinant host cell. Examples of FAR polypeptides
useful in accordance with this embodiment are described in PCT
Publication No. WO2010/062480, which is expressly incorporated by
reference herein.
Fatty alcohol may be produced via an acyl-CoA dependent pathway
utilizing fatty acyl-ACP and fatty acyl-CoA intermediates and an
acyl-CoA independent pathway utilizing fatty acyl-ACP intermediates
but not a fatty acyl-CoA intermediate. In particular embodiments,
the enzyme encoded by the over expressed gene is selected from a
fatty acid synthase, an acyl-ACP thioesterase, a fatty acyl-CoA
synthase and an acetyl-CoA carboxylase. In some embodiments, the
protein encoded by the over expressed gene is endogenous to the
host cell. In other embodiments, the protein encoded by the
overexpressed gene is heterologous to the host cell. Fatty alcohols
are also made in nature by enzymes that are able to reduce various
acyl-ACP or acyl-CoA molecules to the corresponding primary
alcohols. See also, U.S. Patent Publication Nos. 20100105963, and
20110206630 and U.S. Pat. No. 8,097,439, expressly incorporated by
reference herein. As used herein, a recombinant host cell or an
engineered host cell refers to a host cell whose genetic makeup has
been altered relative to the corresponding wild-type host cell, for
example, by deliberate introduction of new genetic elements and/or
deliberate modification of genetic elements naturally present in
the host cell. The offspring of such recombinant host cells also
contain these new and/or modified genetic elements. In any of the
aspects of the disclosure described herein, the host cell can be
selected from the group consisting of a plant cell, insect cell,
fungus cell (e.g., a filamentous fungus, such as Candida sp., or a
budding yeast, such as Saccharomyces sp.), an algal cell and a
bacterial cell. In one preferred embodiment, recombinant host cells
are recombinant microbial cells. Examples of host cells that are
microbial cells, include but are not limited to cells from the
genus Escherichia, Bacillus, Lactobacillus, Zymomonas, Rhodococcus,
Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium,
Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora,
Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium,
Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia, or
Streptomyces. In some embodiments, the host cell is a Gram-positive
bacterial cell. In other embodiments, the host cell is a
Gram-negative bacterial cell. In some embodiments, the host cell is
an E. coli cell. In other embodiments, the host cell is a Bacillus
lentus cell, a Bacillus brevis cell, a Bacillus stearothermophilus
cell, a Bacillus lichenoformis cell, a Bacillus alkalophilus cell,
a Bacillus coagulans cell, a Bacillus circulans cell, a Bacillus
pumilis cell, a Bacillus thuringiensis cell, a Bacillus clausii
cell, a Bacillus megaterium cell, a Bacillus subtilis cell, or a
Bacillus amyloliquefaciens cell. In other embodiments, the host
cell is a Trichoderma koningii cell, a Trichoderma viride cell, a
Trichoderma reesei cell, a Trichoderma longibrachiatum cell, an
Aspergillus awamori cell, an Aspergillus fumigates cell, an
Aspergillus foetidus cell, an Aspergillus nidulans cell, an
Aspergillus niger cell, an Aspergillus oryzae cell, a Humicola
insolens cell, a Humicola lanuginose cell, a Rhodococcus opacus
cell, a Rhizomucor miehei cell, or a Mucor michei cell.
In yet other embodiments, the host cell is a Streptomyces lividans
cell or a Streptomyces murinus cell. In yet other embodiments, the
host cell is an Actinomycetes cell. In some embodiments, the host
cell is a Saccharomyces cerevisiae cell. In some embodiments, the
host cell is a Saccharomyces cerevisiae cell. In other embodiments,
the host cell is a cell from a eukaryotic plant, algae,
cyanobacterium, green-sulfur bacterium, green non-sulfur bacterium,
purple sulfur bacterium, purple non-sulfur bacterium, extremophile,
yeast, fungus, an engineered organism thereof, or a synthetic
organism. In some embodiments, the host cell is light-dependent or
fixes carbon. In some embodiments, the host cell is light-dependent
or fixes carbon. In some embodiments, the host cell has autotrophic
activity. In some embodiments, the host cell has photoautotrophic
activity, such as in the presence of light. In some embodiments,
the host cell is heterotrophic or mixotrophic in the absence of
light. In certain embodiments, the host cell is a cell from
Avabidopsis thaliana, Panicum virgatum, Miscanthus giganteus, Zea
mays, Botryococcuse braunii, Chlamydomonas reinhardtii, Dunaliela
salina, Synechococcus Sp. PCC 7002, Synechococcus Sp. PCC 7942,
Synechocystis Sp. PCC 6803, Thermosynechococcus elongates BP-1,
Chlorobium tepidum, Chlorojlexus auranticus, Chromatiumm vinosum,
Rhodospirillum rubrum, Rhodobacter capsulatus, Rhodopseudomonas
palusris, Clostridium ljungdahlii, Clostridiuthermocellum,
Penicillium chrysogenum, Pichia pastoris, Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Pseudomonasjluorescens, or Zymomonas
mobilis.
Culture and Fermentation of Engineered Host Cells
As used herein, fermentation broadly refers to the conversion of
organic materials into target substances by host cells, for
example, the conversion of a carbon source by recombinant host
cells into fatty acids or derivatives thereof by propagating a
culture of the recombinant host cells in a media comprising the
carbon source. As used herein, conditions permissive for the
production means any conditions that allow a host cell to produce a
desired product, such as a fatty acid or a fatty acid derivative.
Similarly, conditions in which the polynucleotide sequence of a
vector is expressed means any conditions that allow a host cell to
synthesize a polypeptide. Suitable conditions include, for example,
fermentation conditions. Fermentation conditions can comprise many
parameters, including but not limited to temperature ranges, levels
of aeration, feed rates and media composition. Each of these
conditions, individually and in combination, allows the host cell
to grow. Fermentation can be aerobic, anaerobic, or variations
thereof (such as micro-aerobic). Exemplary culture media include
broths or gels. Generally, the medium includes a carbon source that
can be metabolized by a host cell directly. In addition, enzymes
can be used in the medium to facilitate the mobilization (e.g., the
depolymerization of starch or cellulose to fermentable sugars) and
subsequent metabolism of the carbon source. For small scale
production, the engineered host cells can be grown in batches of,
for example, about 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L;
fermented; and induced to express a desired polynucleotide
sequence, such as a polynucleotide sequence encoding a CAR
polypeptide. For large scale production, the engineered host cells
can be grown in batches of about 10 L, 100 L, 1000 L, 10,000 L,
100,000 L, 1,000,000 L or larger; fermented; and induced to express
a desired polynucleotide sequence. Alternatively, large scale
fed-batch fermentation may be carried out.
Fatty Alcohol Compositions
The fatty alcohol compositions described herein are found in the
extracellular environment of the recombinant host cell culture and
can be readily isolated from the culture medium. A fatty alcohol
composition may be secreted by the recombinant host cell,
transported into the extracellular environment or passively
transferred into the extracellular environment of the recombinant
host cell culture. The fatty alcohol composition is isolated from a
recombinant host cell culture using routine methods known in the
art. The disclosure provides compositions produced by engineered or
recombinant host cells (bioproducts) which include one or more
fatty aldehydes and/or fatty alcohols. Although a fatty alcohol
component with a particular chain length and degree of saturation
may constitute the majority of the bioproduct produced by a
cultured engineered or recombinant host cell, the composition
typically includes a mixture of fatty aldehydes and/or fatty
alcohols that vary with respect to chain length and/or degree of
saturation. As used herein, fraction of modern carbon or f.sub.M
has the same meaning as defined by National Institute of Standards
and Technology (NIST) Standard Reference Materials (SRMs 4990B and
4990C, known as oxalic acids standards HOxI and HOxII,
respectively. The fundamental definition relates to 0.95 times the
.sup.14C/.sup.12C isotope ratio HOxI (referenced to AD 1950). This
is roughly equivalent to decay-corrected pre-Industrial Revolution
wood. For the current living biosphere (plant material), f.sub.M is
approximately 1.1.
Bioproducts (e.g., the fatty aldehydes and alcohols produced in
accordance with the present disclosure) comprising biologically
produced organic compounds, and in particular, the fatty aldehydes
and alcohols biologically produced using the fatty acid
biosynthetic pathway herein, have not been produced from renewable
sources and, as such, are new compositions of matter. These new
bioproducts can be distinguished from organic compounds derived
from petrochemical carbon on the basis of dual carbon-isotopic
fingerprinting or .sup.14C dating. Additionally, the specific
source of biosourced carbon (e.g., glucose vs. glycerol) can be
determined by dual carbon-isotopic fingerprinting (see, e.g., U.S.
Pat. No. 7,169,588, which is herein incorporated by reference). The
ability to distinguish bioproducts from petroleum based organic
compounds is beneficial in tracking these materials in commerce.
For example, organic compounds or chemicals comprising both
biologically based and petroleum based carbon isotope profiles may
be distinguished from organic compounds and chemicals made only of
petroleum based materials. Hence, the bioproducts herein can be
followed or tracked in commerce on the basis of their unique carbon
isotope profile. Bioproducts can be distinguished from petroleum
based organic compounds by comparing the stable carbon isotope
ratio (.sup.13C/.sup.12C) in each fuel. The .sup.13C/.sup.12C ratio
in a given bioproduct is a consequence of the .sup.13C/.sup.12C
ratio in atmospheric carbon dioxide at the time the carbon dioxide
is fixed. It also reflects the precise metabolic pathway. Regional
variations also occur. Petroleum, C.sub.3 plants (the broadleaf),
C.sub.4 plants (the grasses), and marine carbonates all show
significant differences in .sup.13C/.sup.12C and the corresponding
.delta..sup.13C values. Furthermore, lipid matter of C.sub.3 and
C.sub.4 plants analyze differently than materials derived from the
carbohydrate components of the same plants as a consequence of the
metabolic pathway. Within the precision of measurement, .sup.13C
shows large variations due to isotopic fractionation effects, the
most significant of which for bioproducts is the photosynthetic
mechanism. The major cause of differences in the carbon isotope
ratio in plants is closely associated with differences in the
pathway of photosynthetic carbon metabolism in the plants,
particularly the reaction occurring during the primary
carboxylation (i.e., the initial fixation of atmospheric CO.sub.2).
Two large classes of vegetation are those that incorporate the
C.sub.3 (or Calvin-Benson) photosynthetic cycle and those that
incorporate the C.sub.4 (or Hatch-Slack) photosynthetic cycle. In
C.sub.3 plants, the primary CO.sub.2 fixation or carboxylation
reaction involves the enzyme ribulose-1,5-diphosphate carboxylase,
and the first stable product is a 3-carbon compound. C.sub.3
plants, such as hardwoods and conifers, are dominant in the
temperate climate zones. In C.sub.4 plants, an additional
carboxylation reaction involving another enzyme,
phosphoenol-pyruvate carboxylase, is the primary carboxylation
reaction. The first stable carbon compound is a 4-carbon acid that
is subsequently decarboxylated. The CO.sub.2 thus released is
refixed by the C.sub.3 cycle. Examples of C.sub.4 plants are
tropical grasses, corn, and sugar cane. Both C.sub.4 and C.sub.3
plants exhibit a range of .sup.13C/.sup.12C isotopic ratios, but
typical values are about -7 to about -13 per mil for C.sub.4 plants
and about -19 to about -27 per mil for C.sub.3 plants (see, e.g.,
Stuiver et al., Radiocarbon 19:355 (1977)). Coal and petroleum fall
generally in this latter range. The .sup.13C measurement scale was
originally defined by a zero set by Pee Dee Belemnite (PDB)
limestone, where values are given in parts per thousand deviations
from this material. The ".delta..sup.13C" values are expressed in
parts per thousand (per mil), abbreviated, .Salinity., and are
calculated as follows:
.delta..sup.13C(.Salinity.)=[(.sup.13C/.sup.12C).sub.sample-(.sup.13C/.su-
p.12C).sub.standard]/(.sup.13C/.sup.12C).sub.standard.times.1000
Since the PDB reference material (RM) has been exhausted, a series
of alternative RMs have been developed in cooperation with the
IAEA, USGS, NIST, and other selected international isotope
laboratories. Notations for the per mil deviations from PDB is
.delta..sup.13C. Measurements are made on CO.sub.2 by high
precision stable ratio mass spectrometry (IRMS) on molecular ions
of masses 44, 45, and 46. The compositions described herein include
bioproducts produced by any of the methods described herein,
including, for example, fatty aldehyde and alcohol products.
Specifically, the bioproduct can have a .delta..sup.13C of about
-28 or greater, about -27 or greater, -20 or greater, -18 or
greater, -15 or greater, -13 or greater, -10 or greater, or -8 or
greater. For example, the bioproduct can have a .delta..sup.13C of
about -30 to about -15, about -27 to about -19, about -25 to about
-21, about -15 to about -5, about -13 to about -7, or about -13 to
about -10. In other instances, the bioproduct can have a
.delta..sup.13C of about -10, -11, -12, or -12.3. Bioproducts,
including the bioproducts produced in accordance with the
disclosure herein, can also be distinguished from petroleum based
organic compounds by comparing the amount of .sup.14C in each
compound. Because .sup.14C has a nuclear half-life of 5730 years,
petroleum based fuels containing "older" carbon can be
distinguished from bioproducts which contain "newer" carbon (see,
e.g., Currie, "Source Apportionment of Atmospheric Particles",
Characterization of Environmental Particles, J. Buffle and H. P.
van Leeuwen, Eds., 1 of Vol. I of the IUPAC Environmental
Analytical Chemistry Series (Lewis Publishers, Inc.) 3-74,
(1992)).
The basic assumption in radiocarbon dating is that the constancy of
.sup.14C concentration in the atmosphere leads to the constancy of
.sup.14C in living organisms. However, because of atmospheric
nuclear testing since 1950 and the burning of fossil fuel since
1850, .sup.14C has acquired a second, geochemical time
characteristic. Its concentration in atmospheric CO.sub.2, and
hence in the living biosphere, approximately doubled at the peak of
nuclear testing, in the mid-1960s. It has since been gradually
returning to the steady-state cosmogenic (atmospheric) baseline
isotope rate (.sup.14C/.sup.12C) of about 1.2.times.10.sup.12, with
an approximate relaxation "half-life" of 7-10 years. (This latter
half-life must not be taken literally; rather, one must use the
detailed atmospheric nuclear input/decay function to trace the
variation of atmospheric and biospheric .sup.14C since the onset of
the nuclear age.) It is this latter biospheric .sup.14C time
characteristic that holds out the promise of annual dating of
recent biospheric carbon. .sup.14C can be measured by accelerator
mass spectrometry (AMS), with results given in units of "fraction
of modern carbon" (f.sub.M). f.sub.M is defined by National
Institute of Standards and Technology (NIST) Standard Reference
Materials (SRMs) 4990B and 4990C. As used herein, fraction of
modern carbon (f.sub.M) has the same meaning as defined by National
Institute of Standards and Technology (NIST) Standard Reference
Materials (SRMs) 4990B and 4990C, known as oxalic acids standards
HOxI and HOxII, respectively. The fundamental definition relates to
0.95 times the .sup.14C/.sup.12C isotope ratio HOxI (referenced to
AD 1950). This is roughly equivalent to decay-corrected
pre-Industrial Revolution wood. For the current living biosphere
(plant material), f.sub.M is approximately 1.1. This is roughly
equivalent to decay-corrected pre-Industrial Revolution wood. For
the current living biosphere (plant material), f.sub.M is
approximately 1.1.
The compositions described herein include bioproducts that can have
an f.sub.M .sup.14C of at least about 1. For example, the
bioproduct of the disclosure can have an f.sub.M .sup.14C of at
least about 1.01, an f.sub.M .sup.14C of about 1 to about 1.5, an
f.sub.M .sup.14C of about 1.04 to about 1.18, or an f.sub.M
.sup.14C of about 1.111 to about 1.124. Another measurement of
.sup.14C is known as the percent of modern carbon (pMC). For an
archaeologist or geologist using .sup.14C dates, AD 1950 equals
"zero years old". This also represents 100 pMC. "Bomb carbon" in
the atmosphere reached almost twice the normal level in 1963 at the
peak of thermo-nuclear weapons. Its distribution within the
atmosphere has been approximated since its appearance, showing
values that are greater than 100 pMC for plants and animals living
since AD 1950. It has gradually decreased over time with today's
value being near 107.5 pMC. This means that a fresh biomass
material, such as corn, would give a .sup.14C signature near 107.5
pMC. Petroleum based compounds will have a pMC value of zero.
Combining fossil carbon with present day carbon will result in a
dilution of the present day pMC content. By presuming 107.5 pMC
represents the .sup.14C content of present day biomass materials
and 0 pMC represents the .sup.14C content of petroleum based
products, the measured pMC value for that material will reflect the
proportions of the two component types. For example, a material
derived 100% from present day soybeans would give a radiocarbon
signature near 107.5 pMC. If that material was diluted 50% with
petroleum based products, it would give a radiocarbon signature of
approximately 54 pMC. A biologically based carbon content is
derived by assigning "100%" equal to 107.5 pMC and "0%" equal to 0
pMC. For example, a sample measuring 99 pMC will give an equivalent
biologically based carbon content of 93%. This value is referred to
as the mean biologically based carbon result and assumes all the
components within the analyzed material originated either from
present day biological material or petroleum based material. A
bioproduct comprising one or more fatty aldehydes or alcohols as
described herein can have a pMC of at least about 50, 60, 70, 75,
80, 85, 90, 95, 96, 97, 98, 99, or 100. In other instances, a
bioproduct described herein can have a pMC of between about 50 and
about 100; about 60 and about 100; about 70 and about 100; about 80
and about 100; about 85 and about 100; about 87 and about 98; or
about 90 and about 95. In yet other instances, a bioproduct
described herein can have a pMC of about 90, 91, 92, 93, 94, or
94.2.
Screening Fatty Alcohol Compositions Produced by Recombinant Host
Cell
To determine if conditions are sufficient to allow expression, a
recombinant host cell comprising a heterologous gene or a modified
native gene is cultured, for example, for about 4, 8, 12, 24, 36,
or 48 hours. During and/or after culturing, samples can be obtained
and analyzed to determine if the fatty alcohol production level
(titer, yield or productivity) is different than that of the
corresponding wild type parental cell which has not been modified.
For example, the medium in which the host cells were grown can be
tested for the presence of a desired product. When testing for the
presence of a product, assays, such as, but not limited to, TLC,
HPLC, GC/FID, GC/MS, LC/MS, MS, can be used. Recombinant host cell
strains can be cultured in small volumes (0.001 L to 1 L) of media
in plates or shake flasks in order to screen for altered fatty
alcohol or fatty species production level. Once candidate strains
or "hits" are identified at small scale, these strains are cultured
in larger volumes (1 L to 1000 L) of media in bioreactors, tanks,
and pilot plants to determine the precise fatty alcohol or fatty
species production level. These large volume culture conditions are
used by those skilled in the art to optimize the culture conditions
to obtain desired fatty alcohol or fatty species production.
Utility of Fatty Aldehyde and Fatty Alcohol Compositions
Aldehydes are used to produce many specialty chemicals. For
example, aldehydes are used to produce polymers, resins (e.g.,
Bakelite), dyes, flavorings, plasticizers, perfumes,
pharmaceuticals, and other chemicals, some of which may be used as
solvents, preservatives, or disinfectants. In addition, certain
natural and synthetic compounds, such as vitamins and hormones, are
aldehydes, and many sugars contain aldehyde groups. Fatty aldehydes
can be converted to fatty alcohols by chemical or enzymatic
reduction. Fatty alcohols have many commercial uses. Worldwide
annual sales of fatty alcohols and their derivatives are in excess
of U.S. $1 billion. The shorter chain fatty alcohols are used in
the cosmetic and food industries as emulsifiers, emollients, and
thickeners. Due to their amphiphilic nature, fatty alcohols behave
as nonionic surfactants, which are useful in personal care and
household products, such as, for example, detergents. In addition,
fatty alcohols are used in waxes, gums, resins, pharmaceutical
salves and lotions, lubricating oil additives, textile antistatic
and finishing agents, plasticizers, cosmetics, industrial solvents,
and solvents for fats. The disclosure also provides a surfactant
composition or a detergent composition comprising a fatty alcohol
produced by any of the methods described herein. One of ordinary
skill in the art will appreciate that, depending upon the intended
purpose of the surfactant or detergent composition, different fatty
alcohols can be produced and used. For example, when the fatty
alcohols described herein are used as a feedstock for surfactant or
detergent production, one of ordinary skill in the art will
appreciate that the characteristics of the fatty alcohol feedstock
will affect the characteristics of the surfactant or detergent
composition produced. Hence, the characteristics of the surfactant
or detergent composition can be selected for by producing
particular fatty alcohols for use as a feedstock. A fatty
alcohol-based surfactant and/or detergent composition described
herein can be mixed with other surfactants and/or detergents well
known in the art. In some embodiments, the mixture can include at
least about 10%, at least about 15%, at least about 20%, at least
about 30%, at least about 40%, at least about 50%, at least about
60%, or a range bounded by any two of the foregoing values, by
weight of the fatty alcohol. In other examples, a surfactant or
detergent composition can be made that includes at least about 5%,
at least about 10%, at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 85%, at least about
90%, at least about 95%, or a range bounded by any two of the
foregoing values, by weight of a fatty alcohol that includes a
carbon chain that is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, or 22 carbons in length. Such surfactant or detergent
compositions also can include at least one additive, such as a
microemulsion or a surfactant or detergent from nonmicrobial
sources such as plant oils or petroleum, which can be present in
the amount of at least about 5%, at least about 10%, at least about
15%, at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 85%, at least about 90%, at least about
95%, or a range bounded by any two of the foregoing values, by
weight of the fatty alcohol. The disclosure is further illustrated
by the following examples. The examples are provided for
illustrative purposes only. They are not to be construed as
limiting the scope or content of the disclosure in any way.
EXAMPLES
Example 1
Production Host Modifications--Attenuation of Acyl-CoA
Dehydrogenase
This example describes the construction of a genetically engineered
host cell wherein the expression of a fatty acid degradation enzyme
is attenuated. The fadE gene of Escherichia coli MG1655 (an E. coli
K strain) was deleted using the Lambda Red (also known as the
Red-Driven Integration) system described by Datsenko et al., Proc.
Natl. Acad. Sci. USA 97: 6640-6645 (2000), with the following
modifications:
The following two primers were used to create the deletion of
fadE:
TABLE-US-00001 Del-fadE- (SEQ ID NO: 9)
F5'-AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAAC
ATATTGATTCCGGGGATCCGTCGACC; and Del-fadE- (SEQ ID NO: 10)
R5'-AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAAC
TTTCCTGTAGGCTGGAGCTGCTTC
The Del-fadE-F and Del-fadE-R primers were used to amplify the
kanamycin resistance (KmR) cassette from plasmid pKD13 (described
by Datsenko et al., supra) by PCR. The PCR product was then used to
transform electrocompetent E. coli MG1655 cells containing pKD46
(described in Datsenko et al., supra) that had been previously
induced with arabinose for 3-4 hours. Following a 3-hour outgrowth
in a super optimal broth with catabolite repression (SOC) medium at
37.degree. C., the cells were plated on Luria agar plates
containing 50 .mu.g/mL of Kanamycin. Resistant colonies were
identified and isolated after an overnight incubation at 37.degree.
C. Disruption of the fadE gene was confirmed by PCR amplification
using primers fadE-L2 and fadE-R1, which were designed to flank the
E. coli fadE gene.
The fadE deletion confirmation primers were:
TABLE-US-00002 fadE-L2 (SEQ ID NO: 11) 5'-CGGGCAGGTGCTATGACCAGGAC;
and fadE-R1 (SEQ ID NO: 12) 5'-CGCGGCGTTGACCGGCAGCCTGG
After the fadE deletion was confirmed, a single colony was used to
remove the KmR marker using the pCP20 plasmid as described by
Datsenko et al., supra. The resulting MG1655 E. coli strain with
the fadE gene deleted and the KmR marker removed was named E. coli
MG1655 .DELTA.fadE, or E. coli MG 1655 D1. Fatty acid derivative
("Total Fatty Species") production by the MG1655 E. coli strain
with the fadE gene deleted was compared to fatty acid derivative
production by E. coli MG1655. Cells were transformed with
production plasmid pDG109
(pCL1920_P.sub.TRC_carBopt_12H08_alrAadp1_fabB[A329G]_fadR) and
fermented in glucose minimal media. The data presented in FIG. 5
shows that deletion of the fadE gene did not affect fatty acid
derivative production.
Example 2
Increased Flux Through the Fatty Acid Synthesis Pathway--Acetyl CoA
Carboxylase Mediated
The main precursors for fatty acid biosynthesis are malonyl-CoA and
acetyl-CoA (FIG. 1). It has been suggested that these precursors
limit the rate of fatty acid biosynthesis (FIG. 2) in E. coli. In
this example, synthetic acc operons [Corynebacterium glutamicum
accABCD (.+-.birA)] were overexpressed and the genetic
modifications led to increased acetyl-coA and malonyl-CoA
production in E. coli. In one approach, in order to increase
malonyl-CoA levels, an acetyl-CoA carboxylase enzyme complex from
Corynebacterium glutamicum (C. glutamicum) was overexpressed in E.
coli. Acetyl-CoA carboxylase (acc) consists of four discrete
subunits, accA, accB, accC and accD (FIG. 3). The advantage of C.
glutamicum acc is that two subunits are expressed as fusion
proteins, accCB and accDA, respectively, which facilitates its
balanced expression. Additionally, C. glutamicum birA, which
biotinylates the accB subunit (FIG. 3) was overexpressed. Example 3
describes co-expression of acc genes together with entire fab
operons.
Example 3
Increased Flux Through the Fatty Acid Synthesis Pathway--iFABs
Fatty Acid Derivative Production:
Strategies to increase the flux through the fatty acid synthesis
pathway in recombinant host cells include both overexpression of
native E. coli fatty acid biosynthesis genes and expression of
exogenous fatty acid biosynthesis genes from different organisms in
E. coli. In this study, fatty acid biosynthesis genes from
different organisms were combined in the genome of E. coli DV2.
Sixteen strains containing iFABs 130-145 were evaluated. The
detailed structure of iFABs 130-145 is presented in iFABs Table 1,
below.
TABLE-US-00003 TABLE 1 Components found in iFABs 130-145.
Abbreviation Full Description St_fabD Salmonella typhimurium fabD
gene nSt_fabH Salmonella typhimurium fabH gene with the native RBS
sSt_fabH Salmonella typhimurium fabH gene with a synthetic RBS
Cac_FabF Clostridium acetobutylicum (ATCC824) fabF gene St_fabG
Salmonella typhimurium fabG gene St_fabA Salmonella typhimurium
fabA gene St_fabZ Salmonella typhimurium fabZ gene BS_fabl Bacillus
subtilis fabl gene BS_FabL Bacillus subtilis fabL gene Vc_FabV
Vibrio chorlerae fabV gene Ec_Fabl Escherichia coli fabl gene
Each "iFAB" included various fab genes in the following order: 1)
an enoyl-ACP reductase (BS_fabI, BS_FabL, Vc_FabV, or Ec_FabI); 2)
a b-ketoacyl-ACP synthetase III (St_fabH); 3) a malonyl-CoA-ACP
transacylase (St_fabD); 4) a b-ketoacyl-ACP reductase (St_fabG); 5)
a 3-hydroxy-acyl-ACP dehydratase (St fabA or St fabZ); 6) a
b-ketoacyl-ACP synthetase II (Cac_fabF). Note that St fabA also has
trans-2, cis-3-decenoyl-ACP isomerase activity (ref) and that
Cac_fabF has b-ketoacyl-ACP synthetase II and b-ketoacyl-ACP
synthetase I activities (Zhu et al., BMC Microbiology 9:119
(2009)). See Table 2, below for the specific composition of iFABs
130-145. See FIGS. 7A and B which provide diagrammatic depiction of
the iFAB138 locus, including a diagram of cat-loxP-T5 promoter
integrated in front of FAB138 (7A); and a diagram of iT5_138
(7B).
TABLE-US-00004 TABLE 2 Composition of iFABs 130-145. Ifab BS_fabI
BS_fabL Vc_fabV Ec_fabI nSt_fabH sSt_fabH St_fabD St_fabG St_- fabA
St_fabZ Cac_fabF Ifab130 1 0 0 0 1 1 1 1 1 0 1 Ifab131 1 0 0 0 1 1
1 1 0 1 1 Ifab132 1 0 0 0 0 0 1 1 1 0 1 Ifab133 1 0 0 0 0 0 1 1 0 1
1 Ifab134 0 1 0 0 1 1 1 1 1 0 1 Ifab135 0 1 0 0 1 1 1 1 0 1 1
Ifab136 0 1 0 0 0 0 1 1 1 0 1 Ifab137 0 1 0 0 0 0 1 1 0 1 1 Ifab138
0 0 1 0 1 1 1 1 1 0 1 Ifab139 0 0 1 0 1 1 1 1 0 1 1 Ifab140 0 0 1 0
0 0 1 1 1 0 1 Ifab141 0 0 1 0 0 0 1 1 0 1 1 Ifab142 0 0 0 1 1 1 1 1
1 0 1 Ifab143 0 0 0 1 1 1 1 1 0 1 1 Ifab144 0 0 0 1 0 0 1 1 1 0 1
Ifab145 0 0 0 1 0 0 1 1 0 1 1
The plasmid pCL_P.sub.trc_tesA was transformed into each of the
strains and a fermentation was run in FA2 media with 20 hours from
induction to harvest at both 32.degree. C. and 37.degree. C. Data
for production of Total Fatty Species from duplicate plate screens
is shown in FIGS. 6A and 6B. From this library screen the best
construct was determined to be DV2 with iFAB138. The iFAB138
construct was transferred into strain D178 to make strain EG149.
This strain was used for further engineering. The sequence of
iFAB138 in the genome of EG149 is presented as SEQ ID NO:13. Table
3 presents the genetic characterization of a number of E. coli
strains into which plasmids containing the expression constructs
described herein were introduced as described below. These strains
and plasmids were used to demonstrate the recombinant host cells,
cultures, and methods of certain embodiments of the present
disclosure. The genetic designations in Table 3 are standard
designations known to those of ordinary skill in the art.
TABLE-US-00005 TABLE 3 Genetic Characterization of E. coli strains
Strain Genetic Characterization DV2 MG1655 F-, .lamda.-, ilvG-,
rfb-50, rph-1, .DELTA.fhuA::FRT, .DELTA.fadE::FRT DV2.1 DV2
fabB::fabB[A329V] D178 DV2.1 entD::FRT_P.sub.T5.sub.--entD EG149
D178 .DELTA.insH-11::P.sub.LACUV5-iFAB138 V642 EG149 rph+ SL313
V642 lacIZ::P.sub.A1.sub.--'tesA/pDG109 V668 V642 ilvG.sup.+ LC397
V668 lacIZ::P.sub.TRC.sub.--'tesA(var)_kan SL571 V668 lacIZ::
P.sub.TRC.sub.--'tesA(var)_FRT LC942 SL571
attTn7::P.sub.TRC.sub.--'tesA(var) DG16 LC942/pLC56 V940
LC397/pV171.1 D851 SL571 yijP::Tn5-cat/pV171.1 Plasmids: pDG109,
pLC56 and pV171.1 are pCL_P.sub.trc.sub.--carB_tesA_alrA_fabB_fadR
operon with variable expression of carB and tesA. iFAB138 is SEQ ID
NO: 13.
Example 4
Increasing the Amount of Free Fatty Acid (FFA) Product by Repairing
the Rph and ilvG Mutations
The ilvG and rph mutations were corrected in this strain resulting
in higher production of FFA. Strains D178, EG149 and V668 (Table 3)
were transformed with pCL_P.sub.trc_tesA. Fermentation was run at
32.degree. C. in FA2 media for 40 hours to compare the FFA
production of strains D178, EG149, and V668 with
pCL_P.sub.trc_tesA. Correcting the rph and ilvG mutations resulted
in a 116% increase in the FFA production of the base strain with
pCL_P.sub.trc_tesA. As seen in FIG. 8, V668/pCL_P.sub.trc_tesA
produces more FFA than the D178/pCL P.sub.trc_tesA, or the
EG149/pCL_P.sub.trc_tesA control. Since FFA is a precursor to the
LS9 products, higher FFA production is a good indicator that the
new strain can produce higher levels of LS9 products. Fermentation
and extraction was run according to a standard FALC fermentation
protocol exemplified by the following.
A frozen cell bank vial of the selected E. coli strain was used to
inoculate 20 mL of LB broth in a 125 mL baffled shake flask
containing spectinomycin antibiotic at a concentration of 115
.mu.g/mL. This shake flask was incubated in an orbital shaker at
32.degree. C. for approximately six hours, then 1.25 mL of the
broth was transferred into 125 mL of low P FA2 seed media (2 g/L
NH.sub.4Cl, 0.5 g/L NaCl, 3 g/L KH.sub.2PO.sub.4, 0.25 g/L
MgSO.sub.4-7H2O, 0.015 g/L mM CaCl.sub.2-2H2O, 30 g/L glucose, 1
mL/L of a trace minerals solution (2 g/L of ZnCl.sub.2.4H.sub.2O, 2
g/L of CaCl.sub.2.6H.sub.2O, 2 g/L of Na.sub.2MoO.sub.4.2H.sub.2O,
1.9 g/L of CuSO.sub.4.5H.sub.2O, 0.5 g/L of H.sub.3BO.sub.3, and 10
mL/L of concentrated HCl), 10 mg/L of ferric citrate, 100 mM of
Bis-Tris buffer (pH 7.0), and 115 .mu.g/mL of spectinomycin), in a
500 mL baffled Erlenmeyer shake flask, and incubated on a shaker
overnight at 32.degree. C. 100 mL of this low P FA2 seed culture
was used to inoculate a 5 L Biostat Aplus bioreactor (Sartorius
BBI), initially containing 1.9 L of sterilized F1 bioreactor
fermentation medium. This medium is initially composed of 3.5 g/L
of KH.sub.2PO.sub.4, 0.5 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5 g/L
of MgSO.sub.4 heptahydrate, 10 g/L of sterile filtered glucose, 80
mg/L ferric citrate, 5 g/L Casamino acids, 10 mL/L of the sterile
filtered trace minerals solution, 1.25 mL/L of a sterile filtered
vitamin solution (0.42 g/L of riboflavin, 5.4 g/L of pantothenic
acid, 6 g/L of niacin, 1.4 g/L of pyridoxine, 0.06 g/L of biotin,
and 0.04 g/L of folic acid), and the spectinomycin at the same
concentration as utilized in the seed media. The pH of the culture
was maintained at 6.9 using 28% w/v ammonia water, the temperature
at 33.degree. C., the aeration rate at 1 lpm (0.5 v/v/m), and the
dissolved oxygen tension at 30% of saturation, utilizing the
agitation loop cascaded to the DO controller and oxygen
supplementation. Foaming was controlled by the automated addition
of a silicone emulsion based antifoam (Dow Corning 1410).
A nutrient feed composed of 3.9 g/L MgSO.sub.4 heptahydrate and 600
g/L glucose was started when the glucose in the initial medium was
almost depleted (approximately 4-6 hours following inoculation)
under an exponential feed rate of 0.3 hr.sup.-1 to a constant
maximal glucose feed rate of 10-12 g/L/hr, based on the nominal
fermentation volume of 2 L. Production of fatty alcohol in the
bioreactor was induced when the culture attained an OD of 5 AU
(approximately 3-4 hours following inoculation) by the addition of
a 1M IPTG stock solution to a final concentration of 1 mM. The
bioreactor was sampled twice per day thereafter, and harvested
approximately 72 hours following inoculation. A 0.5 mL sample of
the well-mixed fermentation broth was transferred into a 15 mL
conical tube (VWR), and thoroughly mixed with 5 mL of butyl
acetate. The tube was inverted several times to mix, then vortexed
vigorously for approximately two minutes. The tube was then
centrifuged for five minutes to separate the organic and aqueous
layers, and a portion of the organic layer transferred into a glass
vial for gas chromatographic analysis.
Example 5
Increased Production of Fatty Alcohol by Transposon
Mutagenesis--yijP
To improve the titer, yield, productivity of fatty alcohol
production by E. coli, transposon mutagenesis and high-throughput
screening was carried out and beneficial mutations were sequenced.
A transposon insertion in the yijP strain was shown to improve the
strain's fatty alcohol yield in both shake flask and fed-batch
fermentations. The SL313 strain produces fatty alcohols. The
genotype of this strain is provided in Table 3. Transposon clones
were then subjected to high-throughput screening to measure
production of fatty alcohols. Briefly, colonies were picked into
deep-well plates containing LB, grown overnight, inoculated into
fresh LB and grown for 3 hours, inoculated into fresh FA2.1 media,
grown for 16 hours, then extracted using butyl acetate. The crude
extract was derivatized with BSTFA
(N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using
GC/FID. Spectinomycin (100 mg/L) was included in all media to
maintain selection of the pDG109 plasmid. Hits were selected by
choosing clones that produced a similar total fatty species as the
control strain SL313, but that had a higher percent of fatty
alcohol species and a lower percent of free fatty acids than the
control. Strain 68F11 was identified as a hit and was validated in
a shake flask fermentation using FA2.1 media. A comparison of
transposon hit 68F11 to control strain SL313 indicated that 68F11
produces a higher percentage of fatty alcohol species than the
control, while both strains produce similar titers of total fatty
species. A single colony of hit 68F11, named LC535, was sequenced
to identify the location of the transposon insertion. Briefly,
genomic DNA was purified from a 10 mL overnight LB culture using
the kit ZR Fungal/Bacterial DNA MiniPrep.TM. (Zymo Research
Corporation, Irvine, Calif.) according to the manufacturer's
instructions. The purified genomic DNA was sequenced outward from
the transposon using primers internal to the transposon:
TABLE-US-00006 DG150 (SEQ ID NO: 14)
5'-GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG-3' DG131 (SEQ ID NO:
15) 5'-GAGCCAATATGCGAGAACACCCGAGAA-3'
Strain LC535 was determined to have a transposon insertion in the
yijP gene (FIG. 18). yijP encodes a conserved inner membrane
protein whose function is unclear. The yijP gene is in an operon
and co-transcribed with the ppc gene, encoding phosphoenolpyruvate
carboxylase, and the yijO gene, encoding a predicted DNA-binding
transcriptional regulator of unknown function. Promoters internal
to the transposon likely have effects on the level and timing of
transcription of yijP, ppc and yijO, and may also have effects on
adjacent genes frwD, pflC, pfld, and argE. Promoters internal to
the transposon cassette are shown in FIG. 18, and may have effects
on adjacent gene expression. Strain LC535 was evaluated in a
fed-batch fermentation on two different dates. Both fermentations
demonstrated that LC535 produced fatty alcohols with a higher yield
than control SL313, and the improvement was 1.3-1.9% absolute yield
based on carbon input. The yijP transposon cassette was further
evaluated in a different strain V940, which produces fatty alcohol
at a higher yield than strain SL313. The yijP::Tn5-cat cassette was
amplified from strain LC535 using primers:
TABLE-US-00007 LC277 (SEQ ID NO: 16)
5'-CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGC AG-3' LC278 (SEQ
ID NO: 17) 5'-GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTATC
CAACG-3'
This linear DNA was electroporated into strain SL571 and integrated
into the chromosome using the lambda red recombination system.
Colonies were screened using primers outside the transposon
region:
TABLE-US-00008 DG407 (SEQ ID NO: 18)
5'-AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG-3' (SEQ ID NO: 19) DH408
5'-ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG-3'
A colony with the correct yijP transposon cassette (FIG. 9) was
transformed with the production plasmid pV171.1 to produce strain
D851. D851 (V940 yijP::Tn5-cat) was tested in a shake-flask
fermentation against isogenic strain V940 that does not contain the
yijP transposon cassette. The result of this fermentation showed
that the yijP transposon cassette confers production of a higher
percent of fatty alcohol by the D851 strain relative to the V940
strain and produces similar titers of total fatty species as the
V940 control strain. Strain D851 was evaluated in a fed-batch
fermentation on two different dates. Data from these fermentations
is shown in Table 4 which illustrates that in 5-liter fed-batch
fermentations, strains with the yijP::Tn5-cat transposon insertion
had an increased total fatty species ("FAS") yield and an increase
in percent fatty alcohol ("FALC"). "Fatty Species" include FALC and
FFA.
TABLE-US-00009 TABLE 4 Effect of yijp transposon insertion on titer
and yield of FAS and FALC Strain FAS Titer FAS Yield Percent FALC
FALC Yield V940 68 g/L 18.70% 95.00% 17.80% D851 70 g/L 19.40%
96.10% 18.60% V940 64 g/L 18.40% 91.90% 16.90% D851 67 g/L 19.00%
94.00% 17.80%
Tank
Fermentation Method:
To assess production of fatty acid esters in tank a glycerol vial
of desired strain was used to inoculate 20 mL LB+spectinomycin in
shake flask and incubated at 32.degree. C. for approximately six
hours. 4 mL of LB culture was used to inoculate 125 mL Low PFA Seed
Media (below), which was then incubated at 32.degree. C. shaker
overnight. 50 mL of the overnight culture was used to inoculate 1 L
of Tank Media. Tanks were run at pH 7.2 and 30.5.degree. C. under
pH stat conditions with a maximum feed rate of 16 g/L/hr (glucose
or methanol).
TABLE-US-00010 TABLE 5 Low P FA Seed Media Component Concentration
NH4Cl 2 g/L NaCl 0.5 g/L KH2PO4 1 g/L MgSO4--7H2O 0.25 g/L
CaCl2--2H2O 0.015 g/L Glucose 20 g/L TM2 Trace Minerals solution 1
mL/L Ferric citrate 10 mg/L Bis Tris buffer (pH 7.0) 100 mM
Spectinomycin 115 mg/L
TABLE-US-00011 TABLE 6 Tank Media Component Concentration (NH4)2SO4
0.5 g/L KH2PO4 3.0 g/L Ferric Citrate 0.034 g/L TM2 Trace Minerals
Solution 10 mL/L Casamino acids 5 g/L Post sterile additions
MgSO4--7H2O 2.2 g/L Trace Vitamins Solution 1.25 mL/L Glucose 5 g/L
Inoculum 50 mL/L
Example 6
Addition of an N-terminal 60 bp Fusion Tag to CarB (CarB60)
There are many ways to increase the solubility, stability,
expression or functionality of a protein. In one approach to
increasing the solubility of CarB, a fusion tag could be cloned
before the gene. In another approach increase the expression of
CarB, the promoter or ribosome binding site (RBS) of the gene could
be altered. In this study, carB (SEQ ID NO: 7) was modified by
addition of an N-terminal 60 bp fusion tag. To generate the
modified protein (referred to herein as "CarB60"), carB was first
cloned into the pET15b vector using primers:
TABLE-US-00012 (SEQ ID NO: 20)
5'-GCAATTCCATATGACGAGCGATGTTCACGA-3'; and (SEQ ID NO: 21)
5'-CCGCTCGAGTAAATCAGACCGAACTCGCG.
The pET15b-carB construct contained 60 nucleotides directly
upstream of the carB gene:
5'-ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAG CCAT
(SEQ ID NO:22)
The fusion tag version of carB was renamed carB60. The
pET15b_carB60 was then digested using restriction enzymes NcoI and
HindIII and subcloned into the pCL1920-derived vector OP80 which
was cut with the same enzymes. This plasmid was transformed into
strain V324 (MG1655 .DELTA.fadE::FRT .DELTA.fhuA::FRT fabB::A329V
entD::T5-entD lacIZ::PTRc-'TesA) to evaluate FALC production.
Strains were fermented according to a standard procedure
(summarized below) and the total fatty species titer and total
fatty alcohol titer were quantified. FIG. 10 shows that CarB60
increases fatty alcohol titers and therefore the CarB60 enzyme has
higher total cellular activity than CarB when expressed from a
multicopy plasmid.
To assess production of fatty alcohols in production strains,
transformants were grown in 2 ml of LB broth supplemented with
antibiotics (100 mg/L) at 37.degree. C. After overnight growth, 40
ul of culture was transferred into 2 ml of fresh LB supplemented
with antibiotics. After 3 hours of growth, 2 ml of culture were
transferred into a 125 mL flask containing 20 ml of M9 medium with
3% glucose supplemented with 20 .mu.l trace mineral solution, 10
.mu.g/L iron citrate, 1 .mu.g/L thiamine, and antibiotics (FA2
media). When the OD.sub.600 of the culture reached 1.0, 1 mM of
IPTG was added to each flask. After 20 hours of growth at
37.degree. C., 400 .mu.L samples from each flask were removed and
fatty alcohols extracted with 400 .mu.L butyl acetate. To further
understand the mechanism of the improved CarB activity, CarB60 was
purified from strain D178 which does not contain `TesA (MG1655
.DELTA.fadE::FRT .DELTA.fhuA::FRT fabB::A329V entD::P.sub.T5-entD).
Briefly, pCL1920 carB60 was transformed into strain D178, which has
been engineered for fatty alcohol production, and fermentation was
carried out at 37.degree. C. in FA-2 medium supplemented with
spectinomycin (100 .mu.g/ml). When the culture OD.sub.600 reached
1.6, cells were induced with 1 mM isopropyl-
-D-thiogalactopyranoside (IPTG) and incubated for an additional 23
h at 37.degree. C. For purification of CarB60, the cells were
harvested by centrifugation for 20 min at 4.degree. C. at 4,500
rpm. Cell paste (10 g) was suspended in 12 ml of BugBuster
MasterMix (Novagen) and protease inhibitor cocktail solution. The
cells were disrupted by French Press and the resulting homogenate
was centrifuged at 10,000 rpm to remove cellular debris. Ni-NTA was
added to the resulting mixture, and the suspension was swirled at
4.degree. C. at 100 rpm for 1 hour on a rotary shaker. The slurry
was poured into a column, and the flow-through was collected. The
Ni-NTA resin was washed with 10 mM imidazole in 50 mM sodium
phosphate buffer pH 8.0 containing 300 mM NaCl, and further washed
with 20 mM imidazole in 50 mM sodium phosphate buffer pH 8.0
containing 300 mM NaCl. The CarB60 protein was eluted with 250 mM
imidazole in 50 mM sodium phosphate buffer pH 8.0 containing 300 mM
NaCl, and analyzed by SDS-PAGE. The protein was dialyzed against
20% (v/v) glycerol in 50 sodium phosphate buffer pH 7.5 yielding
approximately 10 mg of CarB60 per liter of culture. The protein was
flash frozen and stored at -80.degree. C. until needed.
The CarB60 protein was abundantly expressed from a multicopy
plasmid. Additional SDS-PAGE analysis showed that expression of
CarB60 was higher than CarB. The higher expression level of CarB60
suggested that the carB60 gene integrated into the E. coli
chromosome would produce more protein than the carB gene in the
same location. To test this hypothesis, the carB60 gene was
integrated into the E. coli chromosome. Briefly, the carB60 gene
was first amplified from pCL carB60 using forward primer:
TABLE-US-00013 (SEQ ID NO: 23)
5'-ACGGATCCCCGGAATGCGCAACGCAATTAATGTaAGTTAGCGC-3';
and reverse primer:
TABLE-US-00014 (SEQ ID NO: 24)
5'-TGCGTCATCGCCATTGAATTCCTAAATCAGACCGAACTCGCGCAG G-3'.
A second PCR product was amplified from vector pAH56 using forward
primer:
5'-ATTCCGGGGATCCGTCGACC-3' (SEQ ID NO:25); and reverse primer:
5'-AATGGCGATGACGCATCCTCACG-3' (SEQ ID NO:26)
This fragment contains a kanamycin resistance cassette, .lamda.attP
site, and .gamma.R6k origin of replication. The two PCR products
were joined using the InFusion kit (Clontech) to create plasmid
pSL116-126. A fatty alcohol production strain containing an
integrated form of `TesA12H08 and a helper plasmid pINT was
transformed with either pSL116-126 containing the carB60 gene or
plasmid F27 containing the carB gene. These strains were fermented
in FA2 media according to standard procedures for shake-flask
fermentations, as described above. To characterize and quantify the
fatty alcohols and fatty acid esters, gas chromatography ("GC")
coupled with flame ionization ("FID") detection was used. The crude
extract was derivatized with BSTFA
(N,O-bis[Trimethylsilyl]trifluoroacetamide) and analyzed using a
GC/FID. Quantification was carried out by injecting various
concentrations of the appropriate authentic references using the GC
method described above as well as assays including, but not limited
to, gas chromatography (GC), mass spectroscopy (MS), thin layer
chromatography (TLC), high-performance liquid chromatography
(HPLC), liquid chromatography (LC), GC coupled with a flame
ionization detector (GC-FID), GC-MS, and LC-MS, can be used. When
testing for the expression of a polypeptide, techniques such as
Western blotting and dot blotting may be used.
The results of the fermentation after 20 hours are shown in FIG.
11. The total fatty product titers of the two strains are similar
(2.4 g/L total fatty species), however integrated CarB60 converts a
greater fraction of C12 and C14 chain length free fatty acids into
fatty alcohols, compared to CarB without the N-terminal tag. These
data suggest that cells expressing CarB60 have a higher total
cellular carboxylic acid reductase activity, and can convert more
FFA into fatty alcohols. Thus, carB60 when integrated in the
chromosome is an improved carB template that provides desired
activity for evolving carB gene to identity improved carB
variants.
Example 7
Generation of CarB Mutants
The CarB enzyme is a rate-limiting step in the production of fatty
alcohols under certain process conditions. To produce fatty
alcohols economically, efforts were made to increase the activity
of the CarB enzyme.
Error Prone PCR Library Screen:
Random mutagenesis using error prone PCR was performed under
conditions where the copying fidelity of the DNA polymerase is low.
The mutagenized nucleic acids were cloned into a vector, and
error-prone PCR followed by high-throughput screening was done to
find beneficial mutations that increase conversion of free fatty
acids to fatty alcohols (as detailed below). Important residues
were further mutated to other amino acids. A number of single amino
acid mutations and combinations of mutations increased the fraction
of fatty species that are converted to fatty alcohols. Briefly,
random mutations were generated in the carB60opt gene by
error-prone PCR using the Genemorph II kit (Stratagene). Mutations
were generated in only one of two domains of carB60opt separately,
to facilitate cloning. Library 1 contained the first 759 residues
of carB60opt and was generated by error-prone PCR using
primers:
HZ117 5'-ACGGAAAGGAGCTAGCACATGGGCAGCAGCCATCATCAT-3' (SEQ ID NO:27);
and
DG264 5'-GTAAAGGATGGACGGCGGTCACCCGCC-3' (SEQ ID NO:28). The vector
for Library 1 was plasmid pDG115 digested with enzymes NheI and
PshAI. Library 2 contained the last 435 residues of carB60opt and
was generated by error-prone PCR using primers:
TABLE-US-00015 DG263 (SEQ ID NO: 29)
5'-CACGGCGGGTGACCGCCGTCCATCC-3'; and HZ118 (SEQ ID NO: 30)
5'-TTAATTCCGGGGATCCCTAAATCAGACCGAACTCGCGCAGGTC-3'.
The vector for Library 2 was plasmid pDG115 digested with enzymes
PshAI and BamHI. The error-prone inserts were cloned into the
vectors using InFusion Advantage (Clontech) and passaged through
cloning strain NEB Turbo (New England Biolabs). The libraries were
then transformed into strain EG442 (EG149 Tn7::P.sub.TRC-ABR
lacIZ::P.sub.T50-ABR). Error-prone carB60opt clones were then
subjected to high-throughput screening to measure production of
fatty alcohols. Briefly, colonies were picked into deep-well plates
containing LB, grown overnight, inoculated into fresh LB and grown
for 3 hours, inoculated into fresh FA-2.1 media, grown for 16
hours, then extracted using butyl acetate. The crude extract was
derivatized with BSTFA (N,O-bis[Trimethylsilyl]trifluoroacetamide)
and analyzed using a standard GC/FID method. Spectinomycin (100
mg/L) was included in all media to maintain selection of the pDG115
plasmid. Hits were selected by choosing clones that produced a
smaller total free fatty acid titer and a larger total fatty
alcohol titer compared to the control strain. To compare hits from
different fermentation screens, the conversion of free fatty acids
to fatty alcohols was normalized by calculating a normalized free
fatty acid percentage NORM FFA=Mutant Percent FFA/Control Percent
FFA where "Percent FFA" is the total free fatty acid species titer
divided by the total fatty species titer. Hits were subjected to
further verification using shake-flask fermentations, as described
below.
Hits were sequenced to identify the beneficial mutations.
Sequencing was performed by colony PCR of the entire carB60opt gene
using primers
SL59 5'-CAGCCGTTTATTGCCGACTGGATG-3'(SEQ ID NO:31); and
EG479 5'-CTGTTTTATCAGACCGCTTCTGCGTTC-3' (SEQ ID NO:32), and
sequenced using primers internal to the carB60opt enzyme.
The beneficial mutations that improved the CarB60opt enzyme are
shown in Table 7. The normalized free fatty acid (NORM FFA) column
indicates the improvement in the enzyme, with lower values
indicating the best improvement. "Well #" indicates the primary
screening well that this mutation was found in. All residue numbers
refer to the CarB protein sequence, which does not include the 60
bp tag. Mutations indicated with the prefix "Tag:" indication
mutations in the 60 bp/20 residue N-terminal tag.
TABLE-US-00016 TABLE 7 Beneficial Mutations in the CarB Enzyme
Identified During Error-Prone Screening (TAG Mutations Removed)
Well # Norm FFA Missense Mutations Silent Mutations 131B08 70.50%
L799M V810F S927R M1062L A1158V CCG1116CCT F1170I 20C07 71.80%
A535S 65B02 74.70% M930R ACC867ACA 54B10 76.30% L80Q T231M F288L
A418T V530M A541V G677D P712A 67E1 78.20% D750G R827C D986G G1026D
P1149S GCA1031GCT GTC1073GTT 65C08 78.90% V926A ATT941ATA 12C10
80.30% V46I 66E08 80.10% V926A 70F02 80.90% D750G R827C D986G
G1026D P1149S GCA1031GCT GTC1073GTT 07D01 82.40% E20K V191A 66G09
82.40% R827C L1128S ACG780ACA CTG923TTG 25H02 83.50% F288S 06C01
85.10% V46I 06C01 05D02 85.20% T396S CCG477CCT 124E03 86.00% R827C
L1128S ACG780ACA CTG923TTG 17A04 86.20% A574T GCA237GCT ACC676ACT
GCC529GCT 132C08 87.00% M1062T R1080H TTG830TTA TAC834TAT 72C09
87.30% P809L M1062V 10F02 87.70% E636K 71H03 88.10% R827C L1128S
ACG780ACA CTG923TTG 38G04 88.90% D143E A612T GCA181GCG 42F08 90.20%
T90M CTG186CTT 66C04 90.30% L1128S 18C03 90.40% Q473L 12E02 90.60%
D19N S22N R87H L416S CCG167CCA 28809 91.10% E28K H212N Q473L
CCG122CCA ACG178ACA CTG283TTG CTG340CTA ACC401ACT GCA681GCG 103E09
92.20% E936K P1134R CGT829CGG CTG1007CTA 03E09 93.20% M259I 74G11
93.80% I870V S927I S985I I1164F GTG1000GTC 46C01 95.60% D18V
D292N
Saturation Mutagenesis (Combo 1 and 2 Library Generated):
Amino acid positions deemed beneficial for fatty alcohol production
following error-prone PCR were subjected to further mutagenesis.
Primers containing the degenerate nucleotides NNK or NNS were used
to mutate these positions to other amino acids. The resulting
"saturation mutagenesis libraries" were screened as described above
for the error prone libraries, and hits were identified that
further improved fatty alcohol conversion (a smaller total free
fatty acid titer and a larger total fatty alcohol titer compared to
the parent "control" strain). Single amino acid/codon changes in
nine different positions that improve the production of fatty
alcohols are shown in Table 8. Hits were subjected to further
verification using shake-flask fermentations, as described
herein.
TABLE-US-00017 TABLE 8 Beneficial Mutations in the CarB Enzyme
Identified During Amino Acid Saturation Mutagenesis WT WT Mutant
Mutant Norm Amino Acid Codon Amino Acid Codon FFA E20 GAG F TTC
92.20% L CTG 94.50% L TTG 96.20% R CGC 86.50% S TCG 87.40% V GTG
86.00% V GTC 85.30% Y TAC 88.80% V191 GTC A GCC 88.70% S AGT 98.00%
F288 TTT G GGG 70.30% R AGG 77.20% S TCT 85.60% S AGC 79.60% Q473
CAA A GCG 89.50% F TTC 89.10% H CAC 84.10% I ATC 77.20% K AAG
90.30% L CTA 90.10% M ATG 89.00% R AGG 88.00% V GTG 89.20% W TGG
84.50% Y TAC 86.00% A535 GCC A TCC 71.80% R827 CGC A GCC 93.20% C
TGT 87.90% C TGC 83.20% V926 GTT A GCT 78.10% A GCG 66.30% A GCC
69.50% E GAG 65.80% G GGC 78.60% S927 AGC G GGG 77.60% G GGT 79.30%
I ATC 90.80% K AAG 70.70% V GTG 87.90% M930 ATG K AAG 82.30% R CGG
73.80% R AGG 69.80% L1128 TTG A GCG 92.70% G GGG 89.70% K AAG
94.80% M ATG 95.80% P CCG 98.40% R AGG 90.90% R CGG 88.50% S TCG
88.90% T ACG 96.30% V GTG 93.90% W TGG 78.80% Y TAC 87.90%
Amino acid substitutions deemed beneficial to fatty alcohol
production were next combined. PCR was used to amplify parts of the
carBopt gene containing various desired mutations, and the parts
were joined together using a PCR-based method (Horton, R. M., Hunt,
H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. 1989). The carBopt
gene was screened without the 60 bp N-terminal tag. The mutations
combined in this combination library are shown in Table 9.
TABLE-US-00018 TABLE 9 CarB Mutations from the First Combination
Library Mutation Codon E20V GTG E20S TCG E20R CGC V191S AGT F288R
AGG F288S AGC F288G GGG Q473L CTG Q473W TGG Q473Y TAC Q473I ATC
Q473H CAC A535S TCC
To facilitate screening, the resulting CarB combination library was
then integrated into the chromosome of strain V668 at the lacZ
locus. The sequence of the carBopt gene at this locus is presented
as SEQ ID NO:7. The genotype of strain V668 is MG1655
(.DELTA.fadE::FRT .DELTA.fhuA::FRT .DELTA.fabB::A329V
.DELTA.entD::T5-entD .DELTA.insH-11::P.sub.lacUV5fab138 rph+ ilvG+)
(as shown in Table 3 and FIG. 16). The strains were then
transformed with plasmid pVA3, which contains TesA, a catalytically
inactive CarB enzyme CarB[S693A] which destroys the
phosphopantetheine attachment site, and other genes which increase
the production of free fatty acids. The combination library was
screened as described above for the error prone library. V668 with
integrated carB opt (A535S) in the lacZ region and containing pVA3
was used as the control. Hits were selected that increased the
production of fatty alcohols and were subjected to further
verification using shake-flask fermentations, as described in
Example 5. The improved percentage of fatty alcohol production
following shake flask fermentation of recombinant host cells
expressing CarB combination mutants is shown in FIG. 12.
The integrated CarB combination mutants were amplified from the
integrated carB hits by PCR using the primers:
TABLE-US-00019 EG58 (SEQ ID NO: 33) 5'-GCACTCGACCGGAATTATCG; and
EG626 (SEQ ID NO: 34) 5'-GCACTACGCGTACTGTGAGCCAGAG.
These inserts were re-amplified using primers:
TABLE-US-00020 DG243 (SEQ ID NO: 35)
5'-GAGGAATAAACCATGACGAGCGATGTTCACGACGCGACCGACGGC; and (SEQ ID NO:
36) DG210 5'-CTAAATCAGACCGAACTCGCGCAGG.
Using InFusion cloning, the pooled carB mutants were cloned into a
production plasmid, pV869, which was PCR amplified using
primers:
TABLE-US-00021 DG228 (SEQ ID NO: 37)
5'-CATGGTTTATTCCTCCTTATTTAATCGATAC; and DG318 (SEQ ID NO: 38)
5'-TGACCTGCGCGAGTTCGGTCTGATTTAG.
The carB mutant that performed the best in the shake-flask
fermentation plasmid screen (carB2; Table 11) was designated VA101
and the control strain carrying carBopt [A535S] was designated
VA82. See FIG. 13.
Amino acid substitutions in the reduction domain of carB deemed
beneficial to fatty alcohol production were combined with one of
the best carB-L combination library hits, "carB3" (Table 11). PCR
was used to amplify parts of the carBopt gene containing various
desired mutations in Reduction domain, and the parts were joined
together using SOE PCR. The mutations combined in this combination
library are shown in Table 10.
TABLE-US-00022 TABLE 10 CarB Mutations from the Second Combination
Library Mutation Codon R827C TGC R827A GCA V926A GCG V926E GAG
S927K AAG S927G GGG M930K AAG M930R AGG L1128W TGG
The combination library was screened as described above for the
error prone library. V668 with integrated carB3 in the lacZ region
and containing pVA3 was used as a control. Hits were selected that
exhibited increased production of fatty alcohols and were subjected
to further verification using shake-flask fermentations, as
described above. The results of a shake flask fermentation showing
an improved percentage of fatty alcohol production using a further
CarB combination mutation (carB4) is shown in Table 11. A graphic
depiction of the relative conversion efficiency of low copy CarB
variants is presented in FIG. 14. Results reported in Table 11 are
from bioreactor runs carried out under identical conditions.
TABLE-US-00023 TABLE 11 CAR Variants Name Mutation(s) Strain Tank
data Notes carB None = WT (E20V191 F288 Q473) protein is SEQ ID NO:
7 carB60 None + tag V324 carB1 A535S V940 83% FALC; C12/C14 = 3.4
has one copy of 12H08 chromosomal TE carB2 E20R, F288G, Q473I,
A535S LH375 97% FALC; C12/C14 = 3.6 has two copies of 12H08
chromosomal TE carB2 E20R, F288G, Q473I, A535S LH346 96% FALC;
C12/C14 = 3.7 has one copy of 12H08 chromosomal TE carB3 E20R,
F288G, Q473H, A535S L combo library No examples run in bioreactors
to date carB4 E20R, F288G, Q473H, A535S, R combo library (VA-219)
97% FALC; C12/C14 = 3.9 has two copies of 12H08 chromosomal TE
R827A, S927G carA None See, US Patent Pub. protein is SEQ ID NO: 39
No. 20100105963 FadD9 None See, US Patent Pub. protein is SEQ ID
NO: 40 No. 20100105963
The DNA sequences of CarA, FadD9, CarB, and CarB60 are presented
herein as SEQ ID NO: 41, 42, 43 and 44, respectively.
Identification of Additional Beneficial Mutations in CarB Enzyme by
Saturation Mutagenesis:
A dual-plasmid screening system was later developed and validated
to identify improved CarB variants over CarB4 for FALC production.
The dual-plasmid system met the following criteria: 1) Mutant
clones produce high FA titer to provide fatty acid flux in excess
of CarB activity. This is accomplished by transforming a base
strain (V668 with two copies of chromosomal TE) with a plasmid
(pLYC4, pCL1920_P.sub.TRC_carDead_tesA_alrAadp1_fabB[A329G]_fadR)
that carries the FALC operon with a catalytically inactive CarB
enzyme CarB[S693A] to enhance the production of free fatty acids;
2) The screening plasmid with carB mutant template, preferably
smaller than 9-kb, is amenable to saturation mutagenesis procedures
and is compatible for expression with pLYC4; 3) The dynamic range
of CarB activity is tunable. This is achieved by combining a weaker
promoter (P.sub.TRC1) and alternative start codons (GTG or TTG) to
tune CarB4 expression levels. 3) Good plasmid stability, a
toxin/antitoxin module (ccdBA operon) was introduced to maintain
plasmid stability.
Briefly, the screening plasmid pBZ1
(pACYCDuet-1_P.sub.TRC1-carB4GTG_rrnBter_ccdAB) was constructed
from four parts using In-Fusion HD cloning method (Clontech) by
mixing equal molar ratios of four parts (P.sub.TRC1, carB4 with
ATG/TTG/GTG start codons, rrnB T1T2 terminators with ccdAB, and
pACYCDuet-1 vector). The parts (1 to 4) were PCR amplified by the
following primer pairs: (1) P.sub.TRC1-Forward primer 5'
CGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCAAATCCGGCTCGTA
TAATGTGTG-3' (SEQ ID NO:45) and reverse primer
5'-GGTTTATTCCTCCTTATTTAATCGATACAT-3' (SEQ ID NO:46) using pVA232
(pCL1920_P.sub.TRC_carB4_tesA_alrAadp1_fabB[A329G]_fadR) plasmid as
template. (1) carB4 with ATG/TTG/GTG start codons--Forward primer
carB4 ATG 5'ATGTATCGATTAAATAAGGAGGAATAAACCATGGGCACGAGCGATGTTCACGACG
CGAC-3' (SEQ ID NO:47); carB4 GTG
5'ATGTATCGATTAAATAAGGAGGAATAAACCGTGGGCACGAGCGATGTTCACGACG CGAC-3'
(SEQ ID NO:48); and carB4 TTG
5'-ATGTATCGATTAAATAAGGAGGAATAAACCTTGGGCACGAGCGATGTTCACGACGC GAC-3'
(SEQ ID NO:49); and reverse primer carB4 rev
5'-TTCTAAATCAGACCGAACTCGCGCAG-3' (SEQ ID NO:50), using pVA232
plasmid as template. (3) The rrnB T1T2 terminators with
ccdAB--Forward primer rrnB T1T2 term
5'-CTGCGCGAGTTCGGTCTGATTTAGAATTCCTCGAGGATGGTAGTGTGG-3' (SEQ ID
NO:51) and reverse primer ccdAB rev
5'-CAGTCGACATACGAAACGGGAATGCGG-3' (SEQ ID NO:52), using plasmid
pAH008 (pV171 ccdBA operon). (4) The pACYCDuet-1 vector
backbone--Forward primer pACYC vector for 5'
CCGCATTCCCGTTTCGTATGTCGACTGAAACCTCAGGCATTGAGAAGCACACGGTC-3' (SEQ ID
NO:53) and reverse primer pACYC vector rev
5'-CTCATTTCAGAATATTTGCCAGAACCGTTAATTTCCTAATGCAGGAGTCGCATAAG-3' (SEQ
ID NO:54).
The pBZ1 plasmid was co-expressed with pLYC4 in the strain
described above and validated by shake flask and deep-well plate
fermentation. The fermentation conditions were optimized such that
CarB4_GTG template reproducibly have .about.65% FALC conversion in
both fermentation platforms as described in Example 5. Results for
shake flask fermentation are shown in FIG. 15.
Additional sites (18, 19, 22, 28, 80, 87, 90, 143, 212, 231, 259,
292, 396, 416, 418, 530, 541, 574, 612, 636, 677, 712, 750, 799,
809, 810, 870, 936, 985, 986, 1026, 1062, 1080, 1134, 1149, 1158,
1161, 1170) containing mutations in the improved CarB variants
(Table 7) were subjected to full saturation mutagenesis. Primers
containing the degenerate nucleotides NNK or NNS were used to
mutate these positions to other amino acids by a PCR-based method
(Sawano and Miyawaki 2000, Nucl. Acids Res. 28: e78). Saturation
library was constructed using the pBZ1
(pACYCDuet-1_P.sub.TRC1-carB4GTG_rrnBter_ccdAB) plasmid template.
Mutant clones were transformed into NEB Turbo (New England Biolab)
cloning strains and plasmids were isolated and pooled. The pooled
plasmids were then transformed into a V668 based strain carrying
plasmid pLYC4 and the transformants were selected on LB agar plates
supplemented with antibiotics (100 mg/L spectinomycin and 34 mg/L
chloramphenicol).
CarB variants from the saturation library were then screened for
the production of fatty alcohols. Single colonies were picked
directly into 96-well plates according to a modified deep-well
plate fermentation protocol as described in Example 5. Hits were
selected by choosing clones that produced a smaller total free
fatty acid titer and a larger total fatty alcohol titer compared to
the control strain. To compare hits from different fermentation
batches, the conversion of free fatty acids to fatty alcohols was
normalized by calculating a normalized free fatty acid percentage.
The NORM FFA (%) was also used in hits validation as described in
Example 5. NORM FFA (%)=Mutant Percent FFA/Control Percent FFA;
where "Percent FFA" is the total free fatty acid species titer
divided by the total fatty species titer. Hits were subjected to
further validation using shake-flask fermentations as described in
Example 5. The normalized free fatty acid (NORM FFA) column
indicates the improvement in the enzyme, with lower values
indicating the best improvement. "Hit ID" indicates the primary
screening plate well position where the lower NORM FFA phenotype
was found. Hits mutations were identified by sequencing PCR
products amplified from "Hit" containing pBZ1 plasmids using mutant
carB gene-specific primers (BZ1 for 5'-GGATCTCGACGCTCTCCCTT-3' (SEQ
ID NO:55) and BZ12_ccdAB unique primer
5'-TCAAAAACGCCATTAACCTGATGTTCTG-3' (SEQ ID NO:56). The NORM FFA
values and mutations identified in validated hits are summarized in
Table 12.
TABLE-US-00024 TABLE 12 Beneficial Mutations in CarB4 Enzyme
identified During Amino Acid Saturation Mutagenesis WT Amino WT Hit
ID Mutant NORM Acid Codon (Amino Acid) Codon FFA(%) D18 GAT
P10H5(R) AGG 75.5 P684(L) CTG 83.6 P4H11(T) ACG 80.8 P8D11(P) CCG
81.8 S22 AGC P1F3(R) AGG 57.7 P2G9(R) AGG 55.7 P2A7(N) AAC 90
P8D7(G) GGG 82.1 L80 CTG P8H11(R) AGG 87.4 R87 CGT P7D7(G) GGG 85.2
P5D12(E) GAG 89.4 D75G GAT P8F11(A) GCG 87.6 I87G ATT P3A12(L) CTG
76.6
Identification of Novel Variants of CarB Enzyme by Full
Combinatorial Mutagenesis:
A full combinatorial library was constructed to include the
following amino acid residues: 18D, 18R, 22S, 22R, 473H, 473I,
827R, 827C, 870I, 870L, 926V, 926A, 926E, 927S, 927K, 927G, 930M,
930K, 930R, 1128L, and 1128W. Primers containing native and mutant
codons at all positions were designed for library construction by a
PCR-based method (Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J.
K. and Pease, L. R. 1989). Beneficial mutations conserved in CarB2,
CarB3, and CarB4 (20R, 288G, and 535S) were not changed, therefore,
carB2GTG cloned into pBZ1 (modified pBZ1_P.sub.TRC1_carB2GTG_ccdAB)
was used as PCR template. Library construction was completed by
assembling PCR fragments into CarB ORFs containing the above
combinatorial mutations. The mutant CarB ORFs were then cloned into
the pBZ1 backbone by In-Fusion method (Clontech). The In-Fusion
product was precipitated and electroporated directly into the
screening strain carrying plasmid pLYC4. Library screening,
deep-well plate and shake flask fermentation were carried out as
described in Example 5. The activities (NORM FFA normalized by
CarB2, 100%) of CarB mutants with specific combinatorial mutations
are summarized in Table 13. CarB2, CarB4, and CarB5 (CarB4-S22R)
are included as controls. The NORM FFA column indicates the
improvement in CarB enzyme, with lower values indicating the best
improvement. The fold improvement (X-FIOC) of control (CarB2) is
also shown. All mutations listed are relative to the polypeptide
sequence of CarB wt (SEQ ID NO:7). For example, CarB1 has A535S
mutation, and the CarBDead (a catalytically inactive CarB enzyme)
carries S693A mutation which destroys the phosphopantetheine
attachment site.
Novel CarB Variants for Improved Fatty Alcohol Production in
Bioreactors:
The purpose of identifying novel CarB variants listed in Table 13
is to use them for improved fatty alcohol production. The top CarB
variant (P06B6-S3R, E20R, S22R, F288G, Q473H, A535S, R873S, S927G,
M930R, L1128W) from Table 13 carries a spontaneous mutation (wild
type AGC to AGA) at position 3. Both P06B6 CarB variants, namely
CarB7 (amino acid R by AGA at position 3-S3R, E20R, S22R, F288G,
Q473H, A535S, R873S, S927G, M930R, L1128W), and CarB8 (wild type
amino acid S by AGC at position 3-E20R, S22R, F288G, Q473H, A535S,
R873S, S927G, M930R, L1128W) were made and cloned into the low copy
number fatty alcohol production plasmid backbone pCL1920 to
generate the following fatty alcohol operons differing only in
CarB. The translation initiation codon (GTG) for all CarB variants
(CarB2, CarB7, and carB8) was reverted to ATG to maximize
expression.
TABLE-US-00025
pCL1920_P.sub.TRC.sub.--carB2_tesA_alrAadp1_fabB[A329G]_fadR
pCL1920_P.sub.TRC.sub.--carB7_tesA_alrAadp1_fabB[A329G]_fadR
pCL1920_P.sub.TRC.sub.--carB8_tesA_alrAadp1_fabB[A329G]_fadR
The above described plasmids were transformed into a V668 based
strain with one copy of chromosomal TE, and the resulted strains
were screened in bioreactors as described in EXAMPLE 4. The
improvement (measured by % fatty alcohols in the bioreactor
fermentation product) of CarB7 and CarB8 over CarB2 was shown in
FIG. 16. The order of activity is CarB7>CarB8>CarB2. The
position 3 mutation of CarB7 (AGC to an AGA R rare codon) conferred
higher activity than CarB8, in addition, SDS-PAGE analysis of total
soluble proteins revealed higher expression of CarB7 than CarB8 and
CarB2. The expression levels of CarB2 and CarB8 were similar. This
is consistent with the CarB60 data described in EXAMPLE 6, both the
position 3 AGA R rare codon mutation and the CarB60 tag at its
N-terminus can improve CarB expression. It is understood that the
CarB7 and CarB8 will perform better than CarB2 in strains with
increased free fatty acids flux by either engineering the host
strains and/or engineering the other components of the fatty
alcohol production operon.
TABLE-US-00026 TABLE 13 Summary of CarB Variants Identified from
Combinatorial Library in Dual-Plasmid system. Mutants NORM FFA (%)
X-FIOC Mutations P06B6 16.5 6.06 S3R, E20R, S22R, F288G, Q473H,
A535S, R873S, S927G, M930R, L1128W P13A3 23.9 4.18 D18R, E20R,
S22R, F288G, Q473I, A535S, S927G, M930K, L1128W P02A2 26.5 3.77
E20R, S22R, F288G, Q473I, A535S, R827C, V926E, S927K, M930R P05H3
26.7 3.75 D18R, E20R, 288G, Q473I, A535S, R827C, V926E, M930K,
L1128W P10F10 31.9 3.13 E20R, S22R, F288G, Q473H, A535S, R827C,
V926A, S927K, M930R P01C12 34.2 2.92 E20R, S22R, F288G, Q473H,
A535S, R827C P03B1 36.9 2.71 E20R, S22R, F288G, Q473I, A535S,
R827C, M930R P06E4 36.9 2.71 E20R, S22R, F288G, Q473I, A535S,
I870L, S927G, M930R P14C6 37.4 2.67 E20R, S22R, F288G, Q473I,
A535S, I870L, S927G P05F10 40.4 2.48 D18R, E20R, S22R, F288G,
Q473I, A535S, R827C, I870L, V926A, S927G P06C8 40.8 2.45 E20R,
S22R, F288G, Q473H, A535S, R827C, I870L, L1128W P15E4 40.8 2.45
D18R, E20R, S22R, F288G, Q473H, A535S, R827C, I870L, S927G, L1128W
P05H7 40.9 2.44 E20R, S22R, F288G, Q473I, A535S, R827C, I870L,
S927G, L1128W P15A6 41 2.44 E20R, S22R, F288G, Q473I, A535S, R827C,
I870L, S927G, M930K, L1128W P08F5 41.2 2.43 E20R, S22R, F288G,
Q473H, A535S, I870L, S927G, M930K P14C7 41.3 2.42 E20R, F288G,
Q473I, A535S, I870L, M930K P16H10 42.1 2.38 E20R, S22R, F288G,
Q473H, A535S, S927G, M930K, L1128W P16A1 44.1 2.27 D18R, E20R,
S22R, F288G, Q473I, A535S, S927G, L1128W P14H4 44.2 2.26 E20R,
S22R, F288G, Q473I, A535S, R827C, I870L, S927G P15C1 46.5 2.15
D18R, E20R, S22R, F288G, Q473I, A535S, R827C, I870L, S927G, L1128W
P16E5 47.2 2.12 D18R, E20R, S22R, F288G, Q473I, A535S, S927G,
M930R, L1128W P15A3 47.2 2.12 E20R, S22R, F288G, Q473H, A535S,
V926E, S927G, M930R P05A2 52.4 1.91 E20R, S22R, F288G, Q473H,
A535S, R827C, I870L, V926A, L1128W CarB2 100 1 E20R, F288G, Q473I,
A535S CarB4 77.8 1.29 E20R, F288G, Q473H, A535S, R827A, S927G CarB5
48.9 2.04 E20R, S22R, F288G, Q473H, A535S, R827A, S927G CarB1 ND
A535S CarB wt ND SEQ ID NO: 7 CarBDead ND S693A
All methods described herein can be performed in any suitable order
unless otherwise indicated herein or otherwise clearly contradicted
by context. The use of any and all examples, or exemplary language
(e.g., "such as") provided herein, is intended merely to better
illuminate the disclosure and does not pose a limitation on the
scope of the disclosure unless otherwise claimed. No language in
the specification should be construed as indicating any non-claimed
element as essential to the practice of the disclosure. It is to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to be
limiting. Preferred embodiments of this disclosure are described
herein. Variations of those preferred embodiments may become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the disclosure to be practiced otherwise than as specifically
described herein. Accordingly, this disclosure includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the disclosure unless
otherwise indicated herein or otherwise clearly contradicted by
context.
TABLE-US-00027 TABLE 14 Sequences SEQ ID NO Description Sequence 1
cat-loxP-T5 TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTTCTTTATGTAAAAAAAAC
(in front of gtttTAGGATGCATATGGCGGCCGCataacttcgtataGCATACATtatacg
iFAB138) aagttaTCTAGAGTTGCATGCCTGCAGGtccgcttattatcacttattcagg
cgtagcAaccaggcgtttaagggcaccaataactgccttaaaaaaattacgc
cccgccctgccactcatcgcagtactgttgtaattcattaagcattctgccg
acatggaagccatcacaaacggcatgatgaacctgaatcgccagcggcatca
gcaccttgtcgccttgcgtataatatttgcccatggtgaaaacgggggcgaa
gaagttgtccatattggccacgtttaaatcaaaactggtgaaactcacccag
ggattggctgagacgaaaaacatattctcaataaaccctttagggaaatagg
ccaggttttcaccgtaacacgccacatcttgcgaatatatgtgtagaaactg
ccggaaatcgtcgtggtattcactccagagcgatgaaaacgtttcagtttgc
tcatggaaaacggtgtaacaagggtgaacactatcccatatcaccagctcac
cgtctttcattgccatacggaattccggatgagcattcatcaggcgggcaag
aatgtgaataaaggccggataaaacttgtgcttatttttctttacggtcttt
aaaaaggccgtaatatccagctgaacggtctggttataggtacattgagcaa
ctgactgaaatgcctcaaaatgttctttacgatgccattgggatatatcaac
ggtggtatatccagtgatttttttctccattttagcttccttagctcctgaa
aatctcgataactcaaaaaatacgcccggtagtgatcttatttcattatggt
gaaagttggaacctcttacgtgccgatcaacgtctcattttcgccaaaagtt
ggcccagggcttcccggtatcaacagggacaccaggatttatttattctgcg
aagtgatcttccgtcacaggtatttattcGACTCTAGataacttcgtataGC
ATACATTATACGAAGTTATGGATCCAGCTTATCGATACCGTCaaacAAATCA
TAAAAAATTTATTTGCTTTcaggaaaatttttctgTATAATAGATTCAATTG
CGATGACGACGAACACGCACCTGCAGGAGGAGACCAATGATCATCAAACCTA
AAATTCGTGGATTTATC 2 T5 (in front
TTGTCCATCTTTATATAATTTGGGGGTAGGGTGTTCTTTATGTAAAAAAAAC of iFAB138)
gtaTAGGATGCATATGGCGGCCGCataacttcgtataGCATACATTATACGA
AGTTATGGATCCAGCTTATCGATACCGTCaaacAAATCATAAAAAATTTATT
TGCTTTcaggaaaatttttctgTATAATAGATTCAATTGCGATGACGACGAA
CACGCACCTGCAGGAGGAGACCAATGATCATCAAACCTAAAATTCGTGGATT TATC 3 AlrA
MSNHQIRAYAAMQAGEQVVPYQFDAGELKAHQVEVKVEYCGLCHSDLSVINN Acinetobacter
EWQSSVYPAVAGHEIIGTIIALGSEAKGLKLGQRVGIGWTAETCQACDPCIG sp. M-1
GNQVLCTGEKKATIIGHAGGFADKVRAGWQWVIPLPDDLDPESAGPLLCGGI
TVLDPLLKHKIQATHHVGVIGIGGLGHIAIKLLKAWGCEITAFSSNPDKTEE
LKANGADQVVNSRDAQAIKGTRWKLIILSTANGTLNVKAYLNTLAPKGSLHF
LGVTLEPIPVSVGAIMGGAKSVTSSPTGSPLALRQLLQFAARKNIAPQVELF
PMSQLNEAIERLHSGQARYRIVLKADFD 4 AlrAadp1
MATTNVIHAYAAMQAGEALVPYSFDAGELQPHQVEVKVEYCGLCHSDVSVLN
NEWHSSVYPVVAGHEVIGTITQLGSEAKGLKIGQRVGIGWTAESCQACDQCI
SGQQVLCTGENTATIIGHAGGFADKVRAGWQWVIPLPDELDPTSAGPLLCGG
ITVFDPILKHQIQAIHHVAVIGIGGLGHMAIKLLKAWGCEITAFSSNPNKTD
ELKAMGADHVVNSRDDAEIKSQQGKFDLLLSTVNVPLNWNAYLNTLAPNGTF
HFLGVVMEPIPVPVGALLGGAKSLTASPTGSPAALRKLLEFAARKNIAPQIE MY 5 yjgB
atgTCGATGATAAAAAGCTATGCCGCAAAAGAAGCGGGCGGCGAACTGGAAG
TTTATGAGTACGATCCCGGTGAGCTGAGGCCACAAGATGTTGAAGTGCAGGT
GGATTACTGCGGGATCTGCCATTCCGATCTGTCGATGATCGATAACGAATGG
GGATTTTCACAATATCCGCTGGTTGCCGGGCATGAGGTGATTGGGCGCGTGG
TGGCACTCGGGAGCGCCGCGCAGGATAAAGGTTTGCAGGTCGGTCAGCGTGT
CGGGATTGGCTGGACGGCGCGTAGCTGTGGTCACTGCGACGCCTGTATTAGC
GGTAATCAGATCAACTGCGAGCAAGGTGCGGTGCCGACGATTATGAATCGCG
GTGGCTTTGCCGAGAAGTTGCGTGCGGACTGGCAATGGGTGATTCCACTGCC
AGAAAATATTGATATCGAGTCCGCCGGGCCGCTGTTGTGCGGCGGTATCACG
GTCTTTAAACCACTGTTGATGCACCATATCACTGCTACCAGCCGCGTTGGGG
TAATTGGTATTGGCGGGCTGGGGCATATCGCTATAAAACTTCTGCACGCAAT
GGGATGCGAGGTGACAGCCTTTAGTTCTAATCCGGCGAAAGAGCAGGAAGTG
CTGGCGATGGGTGCCGATAAAGTGGTGAATAGCCGCGATCCGCAGGCACTGA
AAGCACTGGCGGGGCAGTTTGATCTCATTATCAACACCGTCAACGTCAGCCT
CGACTGGCAGCCCTATTTTGAGGCGCTGACCTATGGCGGTAATTTCCATACG
GTCGGTGCGGTTCTCACGCCGCTGTCTGTTCCGGCCTTTACGTTAATTGCGG
GCGATCGCAGCGTCTCTGGTTCTGCTACCGGCACGCCTTATGAGCTGCGTAA
GCTGATGCGTTTTGCCGCCCGCAGCAAGGTTGCGCCGACCACCGAACTGTTC
CCGATGTCGAAAATTAACGACGCCATCCAGCATGTGCGCGACGGTAAGGCGC
GTTACCGCGTGGTGTTGAAAGCCGATTTTtga 6 NRRL5646
MAVDSPDERLQRRIAQLFAEDEQVKAARPLEAVSAAVSAPGMRLAQIAATVM CAR
AGYADRPAAGQRAFELNTDDATGRTSLRLLPRFETITYRELWQRVGEVAAAW
HHDPENPLRAGDFVALLGFTSIDYATLDLADIHLGAVTVPLQASAAVSQLIA
ILTETSPRLLASTPEHLDAAVECLLAGTTPERLVVFDYHPEDDDQRAAFESA
RRRLADAGSLVIVETLDAVRARGRDLPAAPLFVPDTDDDPLALLIYTSGSTG
TPKGAMYTNRLAATMWQGNSMLQGNSQRVGINLNYMPMSHIAGRISLFGVLA
RGGTAYFAAKSDMSTLFEDIGLVRPTEIFFVPRVCDMVFQRYQSELDRRSVA
GADLDTLDREVKADLRQNYLGGRFLVAVVGSAPLAAEMKTFMESVLDLPLHD
GYGSTEAGASVLLDNQIQRPPVLDYKLVDVPELGYFRTDRPHPRGELLLKAE
TTIPGYYKRPEVTAEIFDEDGFYKTGDIVAELEHDRLVYVDRRNNVLKLSQG
EFVTVAHLEAVFASSPLIRQIFIYGSSERSYLLAVIVPTDDALRGRDTATLK
SALAESIQRIAKDANLQPYEIPRDFLIETEPFTIANGLLSGIAKLLRPNLKE
RYGAQLEQMYTDLATGQADELLALRREAADLPVLETVSRAAKAMLGVASADM
RPDAHFTDLGGDSLSALSFSNLLHEIFGVEVPVGVVVSPANELRDLANYIEA
ERNSGAKRPTFTSVHGGGSEIRAADLTLDKFIDARTLAAADSIPHAPVPAQT
VLLTGANGYLGRFLCLEWLERLDKTGGTLICVVRGSDAAAARKRLDSAFDSG
DPGLLEHYQQLAARTLEVLAGDIGDPNLGLDDATWQRLAETVDLIVHPAALV
NHVLPYTQLFGPNVVGTAEIVRLAITARRKPVTYLSTVGVADQVDPAEYQED
SDVREMSAVRVVRESYANGYGNSKWAGEVLLREAHDLCGLPVAVFRSDMILA
HSRYAGQLNVQDVFTRLILSLVATGIAPYSFYRTDADGNRQRAHYDGLPADF
TAAAITALGIQATEGFRTYDVLNPYDDGISLDEFVDWLVESGHPIQRITDYS
DWFHRFETAIRALPEKQRQASVLPLLDAYRNPCPAVRGAILPAKEFQAAVQT
AKIGPEQDIPHLSAPLIDKYVSDLELLQLL* 7 carB
MTSDVHDATDGVTETALDDEQSTRRIAELYATDPEFAAAAPLPAVVDAAHKP
GLRLAEILQTLFTGYGDRPALGYRARELATDEGGRTVTRLLPRFDTLTYAQV
WSRVQAVAAALRHNFAQPIYPGDAVATIGFASPDYLTLDLVCAYLGLVSVPL
QHNAPVSRLAPILAEVEPRILTVSAEYLDLAVESVRDVNSVSQLVVFMHPEV
DDHRDALARAREQLAGKGIAVTTLDAIADEGAGLPAEPIYTADHDQRLAMIL
YTSGSTGAPKGAMYTEAMVARLWTMSFITGDPTPVINVNFMPLNHLGGRIPI
STAVQNGGTSYFVPESDMSTLFEDLALVRPTELGLVPRVADMLYQHHLATVD
RLVTQGADELTAEKQAGAELREQVLGGRVITGFVSTAPLAAEMRAFLDITLG
AHIVDGYGLTETGAVTRDGVIVRPPVIDYKLIDVPELGYFSTDKPYPRGELL
VRSQTLTPGYYKRPEVTASVFDRDGYYHTGDVMAETAPDHLVYVDRRNNVLK
LAQGEFVAVANLEAVFSGAALVRQIFVYGNSERSFLLAVVVPTPEALEQYDP
AALKAALADSLQRTARDAELQSYEVPADFIVETEPFSAANGLLSGVGKLLRP
NLKDRYGQRLEQMYADIAATQANQLRELRRAAATQPVIDTLTQAAATILGTG
SEVASDAHFTDLGGDSLSALTLSNLLSDFFGFEVPVGTIVNPATNLAQLAQH
IEAQRTAGDRRPSFTTVHGADATEIRASELTLDKFIDAETLRAAPGLPKVTT
EPRTVLLSGANGWLGRFLTLQWLERLAPVGGTLITIVRGRDDAAARARLTQA
YDTDPELSRRFAELADRHLRVVAGDIGDPNLGLTPEIWHRLAAEVDLVVHPA
ALVNHVLPYRQLFGPNVVGTAEVIKLALTERIKPVTYLSTVSVAMGIPDFEE
DGDIRTVSPVRPLDGGYANGYGNSKWAGEVLLREAHDLCGLPVATFRSDMIL
AHPRYRGQVNVPDMFTRLLLSLLITGVAPRSFYIGDGERPRAHYPGLTVDFV
AEAVTTLGAQQREGYVSYDVMNPHDDGISLDVFVDWLIRAGHPIDRVDDYDD
WVRRFETALTALPEKRRAQTVLPLLHAFRAPQAPLRGAPEPTEVFHAAVRTA
KVGPGDIPHLDEALIDKYIRDLREFGLI* 8 PPTase is
MVDMKTTHTSLPFAGHTLHFVEFDPANFCEQDLLWLPHYAQLQHAGRKRKTE EntD from
HLAGRIAAVYALREYGYKCVPAIGELRQPVWPAEVYGSISHCGTTALAVVSR E. coli
QPIGIDIEEIFSVQTARELTDNIITPAEHERLADCGLAFSLALTLAFSAKES MG1655
AFKASEIQTDAGFLDYQIISWNKQQVIIHRENEMFAVHWQIKEKIVITLCQH D* 9
Del-fadE-F AAAAACAGCAACAATGTGAGCTTTGTTGTAATTATATTGTAAACATATTGAT
TCCGGGGATCCGTCGACC 10 Del-fadE-R
AAACGGAGCCTTTCGGCTCCGTTATTCATTTACGCGGCTTCAACTTTCCTGT
AGGCTGGAGCTGCTTC 11 fadE-L2 CGGGCAGGTGCTATGACCAGGAC 12 fadE-R1
CGCGGCGTTGACCGGCAGCCTGG 13 iFAB138
TGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAGAGAATAGGAACTT (DNA)
CGGAATAGGAACTTCGAACTGCAGGTCGACGGATCCCCGGAATATTTAAATC
ATTTGTACTTTTTGAACAGCAGAGTCGCATTATGGCCACCGAAGCCCAGGCT
GTTGGACAGAACGTAGTTGACTTCTGCATTACGGCCCTCGTTAGGAACGTAA
TCCAGGTCGCATTCCGGATCCGCCTCTTTGTAGCCGATGGTCGGCGGAATGA
AACCCTCTTCAATAGCTTTGGCACAGATAATCGCTTCGACTGCACCGCCAGC
GCCCAGCAGGTGGCCGGTCATGCTCTTGGTGCTAGACACCGGCACTTTGTAG
GCGTATTCACCCAGGACCGTCTTGATCGCTTGGGTTTCGAAGCTGTCATTGT
ACGCCGTGCTCGTACCGTGCGCGTTGATATAGGAAATGTCCTCTGGGCGGAC
ATTATCTTCTTCCATTGCCAGTTTCATTGCACGTGCACCACCTTCACCATTC
GGCGCTGGGCTCGTGATATGATATGCGTCGCAGGTCGCACCATAGCCAACGA
TCTCGGCATAGATTTTGGCACCACGCTTCAGCGCGTGCTCCAACTCTTCCAA
GATAACGATACCGCTGCCCTCGCCCATCACAAAACCGCTGCGATCCTTATCG
AACGGGATGCTGGCGCGCTTCGGGTCCTCAGATTTGGTCACGGCCTTCATCG
AGGCAAAACCCGCCAGGCTCAACGGGGTGATACCTGCTTCGCTACCACCAGA
GATCATAACGTCGCTATAACCAAACTTAATGTTACGGAAGGACTCACCAATG
CTGTTGTTCGCGCTCGCACATGCGGTGACAATGGTCGTGCAAATACCTTTAG
CGCCATAACGAATCGCCAGATTACCGCTTGCCATATTCGCAATGATCATCGG
AATAGTCATAGGGCTCACACGACCCGGACCTTTGGTAATCAGCTTTTCATCC
TGCTTCTCAATGGTGCCGATGCCGCCAATGCCGCTACCAACAATGACGCCGA
AACGATTCTTATCAATCGACTCCAGGTCCAGTTTGCTGTCCTTGATTGCCTC
ATCCGCCGCAACGATCGCAAACTGGCTAAAACGGTCCATACGGTTCGCCTCA
CGCTTGTCGATAAAGTCCTCCGGGGTGAAGTCCTTCACTTCGGCAGCCAGCT
TAACTTTGAAATCGGTTGCGTCAAACGCTTTGATCTTGTCAATGCCACATTT
ACCCTCTTTGATGCTGCACCAGAAGCTATCAGCGTTGTTACCCACCGGCGTC
ACTGCACCAATACCCGTAATGACAACGCGGCGATTCATtttgttgcctcctt
TTAgaacgcggaagtatcctggaacaaaccgactacaaatcgtgtgcggtat
agatcaggcgaccatccaccagaacctcaccgtccgccaggcccatgatcag
gcgacggtttacgatacgtagaaatgaatacgataggtgactttcctggctg
tcggcagaacctggccggtaaatttcacttcgcccacgcccagagcgcggcc
tagccttcgccgcccaaccagcccaggtagaatcccaccaattgccacatag
catccagacccagacaaccgggcatcaccggatcgccgataaagtggcatcc
gaagaaccatagatccggattgatatccagctcggcttcgacatagcctttg
tcgaaattgccgcccgtttcggtcatcttaacgacgcggtccatcatcagca
tgttcggtgcagggagttgcggccctttagcgccaaacagttcaccacgacc
agaggcaagaaggtcttcttttgtataggattcgcgtttatctaccatgttt
tatgtaaaccttaaaaTTAAACCATGTACATTCCGCCGTTGACGTGCAGAGT
CTCACCAGTGATGTAACTCGCTTCGTCAGAGGCTAAAAATGCAACCGCACTG
GCGATTTCCTGAGCGCCGCCGAGGCGACCCGCAGGCACCTGCGCCAGGATAC
CCGCACGCTGATCGTCAGACAGCGCACGCGTCATGTCCGTTTCAATAAAACC
CGGAGCCACAACATTGACAGTAATACCACGGGACGCAACTTCACGCGCCAGT
GATTTACTGAAACCGATCAGGCCCGCTTTCGCCGCAGCGTAGTTTGCCTGAC
CTGCATTTCCCATGGTACCAACCACAGAACCAATAGTGATAATGCGACCACA
ACGCTTTTTCATCATAGCGCGCATTACCGCTTTTGACAGGCGGAAAACGGAT
GATAAGTTGGTTTCGATAATATCGTTCCACTCATCATCTTTCATTCGCATCA
ACAGATTATCACGAGTGATACCGGCATTATTAACCAGGATATCCACTTCACC
AAATTCTGCGCGAATATTTTCCAGAACAGATTCAATAGATGCAGGATCGGTC
ACATTCAACATCAAACCTTTCCCGTTAGCACCTAAATAGTCGCTAATGTTCT
TCGCACCATTTTCACTGGTCGCAGTCCCGATAACTTTCGCGCCGCGGGCAAC
GAGAGTCTCTGCAATTGCGCGGCCTATGCCACGGCTTGCACCAGTCACCAGC
GCAATCTTTCCTTCAAAGCTCATGGTTTTCCTCTTTTATTGCGTAAGTGCCG
CAGACAGCGCCGCCGGCTCGTTCAGCGCCGACGCTGTCAGGGTGTCGACAAT
ACGTTTCGTCAGACCAGTGAGGACTTTACCTGGACCCACTTCATAAAGATGT
TCAACGCCCTGCGCCGCGATAAATTCCACGCTCTTCGTCCACTGTACCGGAT
TGTACAACTGGCGAACCAGCGCATCGCGGATAGCGGCGGCATCGGTTTCACA
TTTCACGTCAACGTTGTTCACTACCGGCACCGTTGGCGCGCTAAAGGTAATT
TTGGCTAATTCAACCGCCAGCTTATCTGCCGCTGGTTTCATCAGCGCGCAGT
GCGACGGTACGCTCACCGGCAGCGGCAGCGCGCGTTTCGCGCCAGCGGCTTT
ACAGGCTGCGCCCGCACGTTCTACCGCCTCTTTATGCCCGGCGATAACCACC
TGTCCCGGCGAGTTAAAGTTAACCGGCGAAACAACCTGCCCTTCGGCAGATT
CTTCACAGGCTTTAGCAATAGAGGCATCATCCAGCCCGATGATCGCAGACAT
GCCGCCAGTGCCTTCCGGAACCGCTTCCTGCATGAATTTACCGCGCATTTCC
ACCAGACGAACGGCATCAGCAAAGTTGATGACGCCAGCGCAAACCAGCGCGG
AATATTCGCCCAGGCTGTGACCTGCCATTAACGCAGGCATTTTACCGCCCTG
CTGCTGCCAAACGCGCCAAAGCGCGACGGAAGCGGTTAATAACGCCGGCTGC
GTCTGCCAGGTTTTATTCAGTTCTTCCGCTGGACCTTGCTGGGTGAGCGCCC
ACAGATCATATCCCAGAGCCGCAGAAGCTTCAGCAAACGTTTCTTCTACGAT
AGGGTAATTTGCCGCCATCTCGGCCAACATCCCAACGCTCTGAGAACCCTGA
CCGGGGAACACAAATGCAAATTGCGTCATGTTTAAATCCTTATACTAGAAAC
GAATCAGCGCGGAGCCCCAGGTGAATCCACCCCCGAAGGCTTCAAGCAATAC
CAGCTGACCGGCTTTAATTCGCCCGTCACGCACGGCTTCATCCAGCGCGCAC
GGCACAGAAGCCGCGGAGGTATTGCCGTGCCTGTCCAGCGTGACGACGACAT
TGTCCATCGACATGCCGAGTTTTTTCGCTGTCGCGCTAATGATACGCAGGTT
AGCCTGATGCGGCACCAGCCAATCGAGTTCTGAGCGATCCAGGTTATTAGCC
GCCAGCGTCTCATCGACAATATGCGCCAGTTCAGTGACCGCCACTTTAAAGA
CTTCATTGCCCGCCATTGTCAGGTAAATCGGGTTATCCGGATTTACGCGATC
GGCATTCGGCAGGGTCAGTAATTCACCGTAACGGCCATCGGCATGAAGATGA
GTGGAGATAATACCCGGTTCTTCAGAAGCGCTCAGTACGGCCGCGCCTGCGC
CATCGCCGAAAATAATGATCGTACCGCGATCGCCAGGATCGCAAGTGCGGGC
TAATACATCGGAACCGACCACCAGCGCGTGTTTAACCGCGCCGGATTTAACG
TACTGGTCGGCGATGCTTAACGCGTAGGTGAAACCTGCGCACGCTGCCGCGA
CATCAAACGCCGGGCAACCTTTAATACCGAGCATACTTTGAATCTGACATGC
CGCGCTTGGAAATGCATGCGTTGCTGATGTGGTAGCCACCACAATCAAGCCA
ATTTGGTCTTTATCGATCCCCGCCATCTCAATCGCGCGATTCGCAGCGGTAA
AGCCCATCGTCGCGACAGTTTCATTCGGCGCGGCGATATGGCGTTTACGAAT
ACCTGTACGAGTGACAATCCACTCGTCAGAGGTCTCAACCATTTTTTCCAGA
TCGGCGTTAGTCCGCACTTGTTCGGGCAGATAGCTGCCAGTACCAATAATCT
TCGTATACATGTACGCTCAGTCACTaaaTTACTCGATATCAATCACATCAAA
TTCGACTTCTGGATTGACGTCAGCATCGTAATCAATGCCTTCAATGCCAAAG
CCAAACAGCTTGATGAACTCTTCTTTGTACATGTCGTAATCGGTCAGCTCAC
GCAGGTTCTCTGTGGTGATTTGTGGCCACAGATCACGGCAGTGCTGCTGAAT
GTCATCACGCAGTTCCCAGTCATCCAAACGCAGACGATTGTGATCATCCACT
TCCGGCGCTGAACCATCT 14 DG150
GCAGTTATTGGTGCCCTTAAACGCCTGGTTGCTACGCCTG 15 DG131
GAGCCAATATGCGAGAACACCCGAGAA 16 LC277
CGCTGAACGTATTGCAGGCCGAGTTGCTGCACCGCTCCCGCCAGGCAG 17 LC278
GGAATTGCCACGGTGCGGCAGGCTCCATACGCGAGGCCAGGTTATCCAACG 18 DG407
AATCACCAGCACTAAAGTGCGCGGTTCGTTACCCG 19 DG408
ATCTGCCGTGGATTGCAGAGTCTATTCAGCTACG 20 Primer1 for
GCAATTCCATATGACGAGCGATGTTCACGA prep of CarB60 21 Primer2 for
CCGCTCGAGTAAATCAGACCGAACTCGCG
prep of CarB60 22 pET15b-
ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCG carb GCAGCCAT
construct (60 nt directly upstream of the carB gene) 23 carB60
ACGGATCCCCGGAATGCGCAACGCAATTAATGTaAGTTAGCGC amplified from
pCL_carB60 forward primer 24 carB60
TGCGTCATCGCCATTGAATTCCTAAATCAGACCGAACTCGCGCAGG amplified from
pCL_carB60 reverse primer 25 carB60 ATTCCGGGGATCCGTCGACC amplified
from pAH56 forward primer 26 carB60 AATGGCGATGACGCATCCTCACG
amplified from pAH56 reverse primer 27 HZ117 primer
ACGGAAAGGAGCTAGCACATGGGCAGCAGCCATCATCAT 28 DG264
GTAAAGGATGGACGGCGGTCACCCGCC primer 29 DG263 primer
CACGGCGGGTGACCGCCGTCCATCC 30 HZ118 primer
TTAATTCCGGGGATCCCTAAATCAGACCGAACTCGCGCAGGTC 31 SL59 primer
CAGCCGTTTATTGCCGACTGGATG 32 EG479 primer
CTGTTTTATCAGACCGCTTCTGCGTTC 33 Primer EG58 GCACTCGACCGGAATTATCG 34
Primer EG626 GCACTACGCGTACTGTGAGCCAGAG 35 Primer DG243
GAGGAATAAACCATGACGAGCGATGTTCACGACGCGACCGACGGC 36 Primer DG210
CTAAATCAGACCGAACTCGCGCAGG 37 Primer DG228
CATGGTTTATTCCTCCTTATTTAATCGATAC 38 Primer DG318
TGACCTGCGCGAGTTCGGTCTGATTTAG 39 carA
MTIETREDRFNRRIDHLFETDPQFAAARPDEAISAAAADPELRLPAAVKQIL (protein)
AGYADRPALGKRAVEFVTDEEGRTTAKLLPRFDTITYRQLAGRIQAVTNAWH
NHPVNAGDRVAILGFTSVDYTTIDIALLELGAVSVPLQTSAPVAQLQPIVAE
TEPKVIASSVDFLADAVALVESGPAPSRLVVFDYSHEVDDQREAFEAAKGKL
AGTGVVVETITDALDRGRSLADAPLYVPDEADPLTLLIYTSGSTGTPKGAMY
PESKTATMWQAGSKARWDETLGVMPSITLNFMPMSHVMGRGILCSTLASGGT
AYFAARSDLSTFLEDLALVRPTQLNFVPRIWDMLFQEYQSRLDNRRAEGSED
RAEAAVLEEVRTQLLGGRFVSALTGSAPISAEMKSWVEDLLDMHLLEGYGST
EAGAVFIDGQIQRPPVIDYKLVDVPDLGYFATDRPYPRGELLVKSEQMFPGY
YKRPEITAEMFDEDGYYRTGDIVAELGPDHLEYLDRRNNVLKLSQGEFVTVS
KLEAVFGDSPLVRQIYVYGNSARSYLLAVVVPTEEALSRWDGDELKSRISDS
LQDAARAAGLQSYEIPRDFLVETTPFTLENGLLTGIRKLARPKLKAHYGERL
EQLYTDLAEGQANELRELRRNGADRPVVETVSRAAVALLGASVTDLRSDAHF
TDLGGDSLSALSFSNLLHEIFDVDVPVGVIVSPATDLAGVAAYIEGELRGSK
RPTYASVHGRDATEVRARDLALGKFIDAKTLSAAPGLPRSGTEIRTVLLTGA
TGFLGRYLALEWLERMDLVDGKVICLVRARSDDEARARLDATFDTGDATLLE
HYRALAADHLEVIAGDKGEADLGLDHDTWQRLADTVDLIVDPAALVNHVLPY
SQMFGPNALGTAELIRIALTTTIKPYVYVSTIGVGQGISPEAFVEDADIREI
SATRRVDDSYANGYGNSKWAGEVLLREAHDWCGLPVSVFRCDMILADTTYSG
QLNLPDMFTRLMLSLVATGIAPGSFYELDADGNRQRAHYDGLPVEFIAEAIS
TIGSQVTDGFETFHVMNPYDDGIGLDEYVDWLIEAGYPVHRVDDYATWLSRF
ETALRALPERQRQASLLPLLHNYQQPSPPVCGAMAPTDRFRAAVQDAKIGPD
KDIPHVTADVIVKYISNLQMLGLL* 40 FadD9
MSINDQRLTRRVEDLYASDAQFAAASPNEAITQAIDQPGVALPQLIRMVMEG (protein)
YADRPALGQRALRFVTDPDSGRTMVELLPRFETITYRELWARAGTLATALSA
EPAIRPGDRVCVLGFNSVDYTTIDIALIRLGAVSVPLQTSAPVTGLRPIVTE
TEPTMIATSIDNLGDAVEVLAGHAPARLVVFDYHGKVDTHREAVEAARARLA
GSVTIDTLAELIERGRALPATPIADSADDALALLIYTSGSTGAPKGAMYRES
QVMSFWRKSSGWFEPSGYPSITLNFMPMSHVGGRQVLYGTLSNGGTAYFVAK
SDLSTLFEDLALVRPTELCFVPRIWDMVFAEFHSEVDRRLVDGADRAALEAQ
VKAELRENVLGGRFVMALTGSAPISAEMTAWVESLLADVHLVEGYGSTEAGM
VLNDGMVRRPAVIDYKLVDVPELGYFGTDQPYPRGELLVKTQTMFPGYYQRP
DVTAEVFDPDGFYRTGDIMAKVGPDQFVYLDRRNNVLKLSQGEFIAVSKLEA
VFGDSPLVRQIFIYGNSARAYPLAVVVPSGDALSRHGIENLKPVISESLQEV
ARAAGLQSYEIPRDFIIETTPFTLENGLLTGIRKLARPQLKKFYGERLERLY
TELADSQSNELRELRQSGPDAPVLPTLCRAAAALLGSTAADVRPDAHFADLG
GDSLSALSLANLLHEIFGVDVPVGVIVSPASDLRALADHIEAARTGVRRPSF
ASIHGRSATEVHASDLTLDKFIDAATLAAAPNLPAPSAQVRTVLLTGATGFL
GRYLALEWLDRMDLVNGKLICLVRARSDEEAQARLDATFDSGDPYLVRHYRE
LGAGRLEVLAGDKGEADLGLDRVTWQRLADTVDLIVDPAALVNHVLPYSQLF
GPNAAGTAELLRLALTGKRKPYIYTSTIAVGEQIPPEAFTEDADIRAISPTR
RIDDSYANGYANSKWAGEVLLREAHEQCGLPVTVFRCDMILADTSYTGQLNL
PDMFTRLMLSLAATGIAPGSFYELDAHGNRQRAHYDGLPVEFVAEAICTLGT
HSPDRFVTYHVMNPYDDGIGLDEFVDWLNSPTSGSGCTIQRIADYGEWLQRF
ETSLRALPDRQRHASLLPLLHNYREPAKPICGSIAPTDQFRAAVQEAKIGPD
KDIPHLTAAIIAKYISNLRLLGLL* 41 carA (DNA)
atgacgatcgaaacgcgcgaagaccgcttcaaccggcgcattgaccacttgt
tcgaaaccgacccgcagttcgccgccgcccgtcccgacgaggcgatcagcgc
ggctgccgccgatccggagttgcgccttcctgccgcggtcaaacagattctg
gccggctatgcggaccgccctgcgctgggcaagcgcgccgtcgagttcgtca
ccgacgaagaaggccgcaccaccgcgaagctcctgccccgcttcgacaccat
cacctaccgtcagctcgcaggccggatccaggccgtgaccaatgcctggcac
aaccatccggtgaatgccggtgaccgcgtggccatcctgggtacaccagtgt
cgactacacgacgatcgacatcgccctgctcgaactcggcgccgtgtccgta
ccgctgcagaccagtgcgccggtggcccaactgcagccgatcgtcgccgaga
ccgagcccaaggtgatcgcgtcgagcgtcgacttcctcgccgacgcagtcgc
tctcgtcgagtccgggcccgcgccgtcgcgactggtggtgttcgactacagc
cacgaggtcgacgatcagcgtgaggcgttcgaggcggccaagggcaagctcg
caggcaccggcgtcgtcgtcgagacgatcaccgacgcactggaccgcgggcg
gtcactcgccgacgcaccgctctacgtgcccgacgaggccgacccgctgacc
cttctcatctacacctccggcagcaccggcactcccaagggcgcgatgtacc
ccgagtccaagaccgccacgatgtggcaggccgggtccaaggcccggtggga
cgagaccctcggcgtgatgccgtcgatcaccctgaacttcatgcccatgagt
cacgtcatggggcgcggcatcctgtgcagcacactcgccagcggcggaaccg
cgtacttcgccgcacgcagcgacctgtccaccttcctggaggacctcgccct
cgtgcggcccacgcagctcaacttcgttcctcgcatctgggacatgctgttc
caggagtaccagagccgcctcgacaaccgccgcgccgagggatccgaggacc
gagccgaagccgcagtcctcgaagaggtccgcacccaactgctcggcgggcg
attcgtttcggccctgaccggatcggctcccatctcggcggagatgaagagc
tgggtcgaggacctgctcgacatgcatctgctggagggctacggctccaccg
aggccggcgcggtgttcatcgacgggcagatccagcgcccgccggtcatcga
ctacaagctggtcgacgtgcccgatctcggctacttcgccacggaccggccc
tacccgcgcggcgaacttctggtcaagtccgagcagatgttccccggctact
acaagcgtccggagatcaccgccgagatgttcgacgaggacgggtactaccg
caccggcgacatcgtcgccgagctcgggcccgaccatctcgaatacctcgac
cgccgcaacaacgtgctgaaactgtcgcagggcgaattcgtcacggtctcca
agctggaggcggtgttcggcgacagccccctggtacgccagatctacgtcta
cggcaacagcgcgcggtcctatctgctggcggtcgtggtcccgaccgaagag
gcactgtcacgttgggacggtgacgaactcaagtcgcgcatcagcgactcac
tgcaggacgcggcacgagccgccggattgcagtcgtatgagatcccgcgtga
cttcctcgtcgagacaacacctacacgctggagaacggcctgctgaccggta
tccgcaagctggcccggccgaaactgaaggcgcactacggcgaacgcctcga
acagctctacaccgacctggccgaggggcaggccaacgagttgcgcgagttg
cgccgcaacggagccgaccggcccgtggtcgagaccgtcagccgcgccgcgg
tcgcactgctcggtgcctccgtcacggatctgcggtccgatgcgcacttcac
cgatctgggtggagattcgttgtcggccttgagcttctcgaacctgttgcac
gagatcttcgatgtcgacgtgccggtcggcgtcatcgtcagcccggccaccg
acctggcaggcgtcgcggcctacatcgagggcgaactgcgcggctccaagcg
ccccacatacgcgtcggtgcacgggcgcgacgccaccgaggtgcgcgcgcgt
gatctcgccctgggcaagttcatcgacgccaagaccctgtccgccgcgccgg
gtctgccgcgttcgggcaccgagatccgcaccgtgctgctgaccggcgccac
cgggttcctgggccgctatctggcgctggaatggctggagcgcatggacctg
gtggacggcaaggtgatctgcctggtgcgcgcccgcagcgacgacgaggccc
gggcgcgtctggacgccacgttcgacaccggggacgcgacactgctcgagca
ctaccgcgcgctggcagccgatcacctcgaggtgatcgccggtgacaagggc
gaggccgatctgggtctcgaccacgacacgtggcagcgactggccgacaccg
tcgatctgatcgtcgatccggccgccctggtcaatcacgtcctgccgtacag
ccagatgttcggacccaatgcgctcggcaccgccgaactcatccggatcgcg
ctgaccaccacgatcaagccgtacgtgtacgtctcgacgatcggtgtgggac
agggcatctcccccgaggcgttcgtcgaggacgccgacatccgcgagatcag
cgcgacgcgccgggtcgacgactcgtacgccaacggctacggcaacagcaag
tgggccggcgaggtcctgctgcgggaggcgcacgactggtgtggtctgccgg
tctcggtgttccgctgcgacatgatcctggccgacacgacctactcgggtca
gctgaacctgccggacatgttcacccgcctgatgctgagcctcgtggcgacc
ggcatcgcgcccggttcgttctacgaactcgatgcggacggcaaccggcagc
gcgcccactacgacgggctgcccgtggagttcatcgccgaggcgatctccac
catcggctcgcaggtcaccgacggattcgagacgttccacgtgatgaacccg
tacgacgacggcatcggcctcgacgagtacgtggactggctgatcgaggccg
gctaccccgtgcaccgcgtcgacgactacgccacctggctgagccggttcga
aaccgcactgcgggccctgccggaacggcaacgtcaggcctcgctgctgccg
ctgctgcacaactatcagcagccctcaccgcccgtgtgcggtgccatggcac
ccaccgaccggttccgtgccgcggtgcaggacgcgaagatcggccccgacaa
ggacattccgcacgtcacggccgacgtgatcgtcaagtacatcagcaacctg
cagatgctcggattgctgtaa 42 FadD9 (DNA)
atgtcgatcaacgatcagcgactgacacgccgcgtcgaggacctatacgcca
gcgacgcccagttcgccgccgccagtcccaacgaggcgatcacccaggcgat
cgaccagcccggggtcgcgcttccacagctcatccgtatggtcatggagggc
tacgccgatcggccggcactcggccagcgtgcgctccgcttcgtcaccgacc
ccgacagcggccgcaccatggtcgagctactgccgcggttcgagaccatcac
ctaccgcgaactgtgggcccgcgccggcacattggccaccgcgttgagcgct
gagcccgcgatccggccgggcgaccgggtttgcgtgctgggcttcaacagcg
tcgactacacaaccatcgacatcgcgctgatccggttgggcgccgtgtcggt
tccactgcagaccagtgcgccggtcaccgggttgcgcccgatcgtcaccgag
accgagccgacgatgatcgccaccagcatcgacaatcttggcgacgccgtcg
aagtgctggccggtcacgccccggcccggctggtcgtattcgattaccacgg
caaggttgacacccaccgcgaggccgtcgaagccgcccgagctcggttggcc
ggctcggtgaccatcgacacacttgccgaactgatcgaacgcggcagggcgc
tgccggccacacccattgccgacagcgccgacgacgcgctggcgctgctgat
ttacacctcgggtagtaccggcgcacccaaaggcgccatgtatcgcgagagc
caggtgatgagcttctggcgcaagtcgagtggctggttcgagccgagcggtt
acccctcgatcacgctgaacttcatgccgatgagccacgtcgggggccgtca
ggtgctctacgggacgctttccaacggcggtaccgcctacttcgtcgccaag
agcgacctgtcgacgctgttcgaggacctcgccctggtgcggcccacagaat
tgtgcttcgtgccgcgcatctgggacatggtgttcgcagagttccacagcga
ggtcgaccgccgcttggtggacggcgccgatcgagcggcgctggaagcgcag
gtgaaggccgagctgcgggagaacgtgctcggcggacggtttgtcatggcgc
tgaccggttccgcgccgatctccgctgagatgacggcgtgggtcgagtccct
gctggccgacgtgcatttggtggagggttacggctccaccgaggccgggatg
gtcctgaacgacggcatggtgcggcgccccgcggtgatcgactacaagctgg
tcgacgtgcccgagctgggctacttcggcaccgatcagccctacccccgggg
cgagctgctggtcaagacgcaaaccatgttccccggctactaccagcgcccg
gatgtcaccgccgaggtgttcgaccccgacggcttctaccggaccggggaca
tcatggccaaagtaggccccgaccagttcgtctacctcgaccgccgcaacaa
cgtgctaaagctctcccagggcgagttcatcgccgtgtcgaagctcgaggcg
gtgttcggcgacagcccgctggtccgacagatcttcatctacggcaacagtg
cccgggcctacccgctggcggtggttgtcccgtccggggacgcgctttctcg
ccatggcatcgagaatctcaagcccgtgatcagcgagtccctgcaggaggta
gcgagggcggccggcctgcaatcctacgagattccacgcgacttcatcatcg
aaaccacgccgttcaccctggagaacggcctgctcaccggcatccgcaagct
ggcacgcccgcagttgaagaagttctatggcgaacgtctcgagcggctctat
accgagctggccgatagccaatccaacgagctgcgcgagctgcggcaaagcg
gtcccgatgcgccggtgcttccgacgctgtgccgtgccgcggctgcgttgct
gggctctaccgctgcggatgtgcggccggacgcgcacttcgccgacctgggt
ggtgactcgctctcggcgctgtcgttggccaacctgctgcacgagatcttcg
gcgtcgacgtgccggtgggtgtcattgtcagcccggcaagcgacctgcgggc
cctggccgaccacatcgaagcagcgcgcaccggcgtcaggcgacccagcttc
gcctcgatacacggtcgctccgcgacggaagtgcacgccagcgacctcacgc
tggacaagttcatcgacgctgccaccctggccgcagccccgaacctgccggc
accgagcgcccaagtgcgcaccgtactgctgaccggcgccaccggctttttg
ggtcgctacctggcgctggaatggctcgaccgcatggacctggtcaacggca
agctgatctgcctggtccgcgccagatccgacgaggaagcacaagcccggct
ggacgcgacgttcgatagcggcgacccgtatttggtgcggcactaccgcgaa
ttgggcgccggccgcctcgaggtgctcgccggcgacaagggcgaggccgacc
tgggcctggaccgggtcacctggcagcggctagccgacacggtggacctgat
cgtggaccccgcggccctggtcaaccacgtgctgccgtatagccagctgttc
ggcccaaacgcggcgggcaccgccgagttgcttcggctggcgctgaccggca
agcgcaagccatacatctacacctcgacgatcgccgtgggcgagcagatccc
gccggaggcgttcaccgaggacgccgacatccgggccatcagcccgacccgc
aggatcgacgacagctacgccaacggctacgcgaacagcaagtgggccggcg
aggtgctgctgcgcgaagctcacgagcagtgcggcctgccggtgacggtctt
ccgctgcgacatgatcctggccgacaccagctataccggtcagctcaacctg
ccggacatgttcacccggctgatgctgagcctggccgctaccggcatcgcac
ccggttcgttctatgagctggatgcgcacggcaatcggcaacgcgcccacta
tgacggcttgccggtcgaattcgtcgcagaagccatttgcacccttgggaca
catagcccggaccgttttgtcacctaccacgtgatgaacccctacgacgacg
gcatcgggctggacgagttcgtcgactggctcaactccccaactagcgggtc
cggttgcacgatccagcggatcgccgactacggcgagtggctgcagcggttc
gagacttcgctgcgtgccttgccggatcgccagcgccacgcctcgctgctgc
ccttgctgcacaactaccgagagcctgcaaagccgatatgcgggtcaatcgc
gcccaccgaccagttccgcgctgccgtccaagaagcgaaaatcggtccggac
aaagacattccgcacctcacggcggcgatcatcgcgaagtacatcagcaacc
tgcgactgctcgggctgctgtga 43 carB (DNA)
atgaccagcgatgttcacgacgccacagacggcgtcaccgaaaccgcactcg
acgacgagcagtcgacccgccgcatcgccgagctgtacgccaccgatcccga
gttcgccgccgccgcaccgttgcccgccgtggtcgacgcggcgcacaaaccc
gggctgcggctggcagagatcctgcagaccctgttcaccggctacggtgacc
gcccggcgctgggataccgcgcccgtgaactggccaccgacgagggcgggcg
caccgtgacgcgtctgctgccgcggttcgacaccctcacctacgcccaggtg
tggtcgcgcgtgcaagcggtcgccgcggccctgcgccacaacttcgcgcagc
cgatctaccccggcgacgccgtcgcgacgatcggtacgcgagtcccgattac
ctgacgctggatctcgtatgcgcctacctgggcctcgtgagtgttccgctgc
agcacaacgcaccggtcagccggctcgccccgatcctggccgaggtcgaacc
gcggatcctcaccgtgagcgccgaatacctcgacctcgcagtcgaatccgtg
cgggacgtcaactcggtgtcgcagctcgtggtgttcgaccatcaccccgagg
tcgacgaccaccgcgacgcactggcccgcgcgcgtgaacaactcgccggcaa
gggcatcgccgtcaccaccctggacgcgatcgccgacgagggcgccgggctg
ccggccgaaccgatctacaccgccgaccatgatcagcgcctcgcgatgatcc
tgtacacctcgggttccaccggcgcacccaagggtgcgatgtacaccgaggc
gatggtggcgcggctgtggaccatgtcgttcatcacgggtgaccccacgccg
gtcatcaacgtcaacttcatgccgctcaaccacctgggcgggcgcatcccca
taccaccgccgtgcagaacggtggaaccagttacttcgtaccggaatccgac
atgtccacgctgttcgaggatctcgcgctggtgcgcccgaccgaactcggcc
tggttccgcgcgtcgccgacatgctctaccagcaccacctcgccaccgtcga
ccgcctggtcacgcagggcgccgacgaactgaccgccgagaagcaggccggt
gccgaactgcgtgagcaggtgctcggcggacgcgtgatcaccggattcgtca
gcaccgcaccgctggccgcggagatgagggcgttcctcgacatcaccctggg
cgcacacatcgtcgacggctacgggctcaccgagaccggcgccgtgacacgc
gacggtgtgatcgtgcggccaccggtgatcgactacaagctgatcgacgttc
ccgaactcggctacttcagcaccgacaagccctacccgcgtggcgaactgct
ggtcaggtcgcaaacgctgactcccgggtactacaagcgccccgaggtcacc
gcgagcgtcttcgaccgggacggctactaccacaccggcgacgtcatggccg
agaccgcacccgaccacctggtgtacgtggaccgtcgcaacaacgtcctcaa
actcgcgcagggcgagttcgtggcggtcgccaacctggaggcggtgttctcc
ggcgcggcgctggtgcgccagatcttcgtgtacggcaacagcgagcgcagta
ccttctggccgtggtggtcccgacgccggaggcgctcgagcagtacgatccg
gccgcgctcaaggccgcgctggccgactcgctgcagcgcaccgcacgcgacg
ccgaactgcaatcctacgaggtgccggccgatttcatcgtcgagaccgagcc
gttcagcgccgccaacgggctgctgtcgggtgtcggaaaactgctgcggccc
aacctcaaagaccgctacgggcagcgcctggagcagatgtacgccgatatcg
cggccacgcaggccaaccagttgcgcgaactgcggcgcgcggccgccacaca
accggtgatcgacaccctcacccaggccgctgccacgatcctcggcaccggg
agcgaggtggcatccgacgcccacttcaccgacctgggcggggattccctgt
cggcgctgacactacgaacctgctgagcgatacttcggtttcgaagttcccg
tcggcaccatcgtgaacccggccaccaacctcgcccaactcgcccagcacat
cgaggcgcagcgcaccgcgggtgaccgcaggccgagtacaccaccgtgcacg
gcgcggacgccaccgagatccgggcgagtgagctgaccctggacaagttcat
cgacgccgaaacgctccgggccgcaccgggtctgcccaaggtcaccaccgag
ccacggacggtgttgctctcgggcgccaacggctggctgggccggttcctca
cgttgcagtggctggaacgcctggcacctgtcggcggcaccctcatcacgat
cgtgcggggccgcgacgacgccgcggcccgcgcacggctgacccaggcctac
gacaccgatcccgagttgtcccgccgcttcgccgagctggccgaccgccacc
tgcgggtggtcgccggtgacatcggcgacccgaatctgggcctcacacccga
gatctggcaccggctcgccgccgaggtcgacctggtggtgcatccggcagcg
ctggtcaaccacgtgctcccctaccggcagctgttcggccccaacgtcgtgg
gcacggccgaggtgatcaagctggccctcaccgaacggatcaagcccgtcac
gtacctgtccaccgtgtcggtggccatggggatccccgacttcgaggaggac
ggcgacatccggaccgtgagcccggtgcgcccgctcgacggcggatacgcca
acggctacggcaacagcaagtgggccggcgaggtgctgctgcgggaggccca
cgatctgtgcgggctgcccgtggcgacgttccgctcggacatgatcctggcg
catccgcgctaccgcggtcaggtcaacgtgccagacatgttcacgcgactcc
tgttgagcctcttgatcaccggcgtcgcgccgcggtcgttctacatcggaga
cggtgagcgcccgcgggcgcactaccccggcctgacggtcgatttcgtggcc
gaggcggtcacgacgctcggcgcgcagcagcgcgagggatacgtgtcctacg
acgtgatgaacccgcacgacgacgggatctccctggatgtgttcgtggactg
gctgatccgggcgggccatccgatcgaccgggtcgacgactacgacgactgg
gtgcgtcggttcgagaccgcgttgaccgcgcttcccgagaagcgccgcgcac
agaccgtactgccgctgctgcacgcgttccgcgctccgcaggcaccgttgcg
cggcgcacccgaacccacggaggtgttccacgccgcggtgcgcaccgcgaag
gtgggcccgggagacatcccgcacctcgacgaggcgctgatcgacaagtaca
tacgcgatctgcgtgagttcggtctgatctga 44 carB60
atgggcagcagccatcatcatcatcatcacagcagcggcctggtgccgcgcg (DNA)
gcagccatATGACGAGCGATGTTCACGACGCGACCGACGGCGTTACCGAGAC
TGCACTGGATGATGAGCAGAGCACTCGTCGTATTGCAGAACTGTACGCAACG
GACCCAGAGTTCGCAGCAGCAGCTCCTCTGCCGGCCGTTGTCGATGCGGCGC
ACAAACCGGGCCTGCGTCTGGCGGAAATCCTGCAGACCCTGTTCACCGGCTA
CGGCGATCGTCCGGCGCTGGGCTATCGTGCACGTGAGCTGGCGACGGACGAA
GGCGGTCGTACGGTCACGCGTCTGCTGCCGCGCTTCGATACCCTGACCTATG
CACAGGTGTGGAGCCGTGTTCAAGCAGTGGCTGCAGCGTTGCGTCACAATTT
CGCACAACCGATTTACCCGGGCGACGCGGTCGCGACTATCGGCTTTGCGAGC
CCGGACTATTTGACGCTGGATCTGGTGTGCGCGTATCTGGGCCTGGTCAGCG
TTCCTTTGCAGCATAACGCTCCGGTGTCTCGCCTGGCCCCGATTCTGGCCGA
GGTGGAACCGCGTATTCTGACGGTGAGCGCAGAATACCTGGACCTGGCGGTT
GAATCCGTCCGTGATGTGAACTCCGTCAGCCAGCTGGTTGTTTTCGACCATC
ATCCGGAAGTGGACGATCACCGTGACGCACTGGCTCGCGCACGCGAGCAGCT
GGCCGGCAAAGGTATCGCAGTTACGACCCTGGATGCGATCGCAGACGAAGGC
GCAGGTTTGCCGGCTGAGCCGATTTACACGGCGGATCACGATCAGCGTCTGG
CCATGATTCTGTATACCAGCGGCTCTACGGGTGCTCCGAAAGGCGCGATGTA
CACCGAAGCGATGGTGGCTCGCCTGTGGACTATGAGCTTTATCACGGGCGAC
CCGACCCCGGTTATCAACGTGAACTTCATGCCGCTGAACCATCTGGGCGGTC
GTATCCCGATTAGCACCGCCGTGCAGAATGGCGGTACCAGCTACTTCGTTCC
GGAAAGCGACATGAGCACGCTGTTTGAGGATCTGGCCCTGGTCCGCCCTACC
GAACTGGGTCTGGTGCCGCGTGTTGCGGACATGCTGTACCAGCATCATCTGG
CGACCGTGGATCGCCTGGTGACCCAGGGCGCGGACGAACTGACTGCGGAAAA
GCAGGCCGGTGCGGAACTGCGTGAACAGGTCTTGGGCGGTCGTGTTATCACC
GGTTTTGTTTCCACCGCGCCGTTGGCGGCAGAGATGCGTGCTTTTCTGGATA
TCACCTTGGGTGCACACATCGTTGACGGTTACGGTCTGACCGAAACCGGTGC
GGTCACCCGTGATGGTGTGATTGTTCGTCCTCCGGTCATTGATTACAAGCTG
ATCGATGTGCCGGAGCTGGGTTACTTCTCCACCGACAAACCGTACCCGCGTG
GCGAGCTGCTGGTTCGTAGCCAAACGTTGACTCCGGGTTACTACAAGCGCCC
AGAAGTCACCGCGTCCGTTTTCGATCGCGACGGCTATTACCACACCGGCGAC
GTGATGGCAGAAACCGCGCCAGACCACCTGGTGTATGTGGACCGCCGCAACA
ATGTTCTGAAGCTGGCGCAAGGTGAATTTGTCGCCGTGGCTAACCTGGAGGC
CGTTTTCAGCGGCGCTGCTCTGGTCCGCCAGATTTTCGTGTATGGTAACAGC
GAGCGCAGCTTTCTGTTGGCTGTTGTTGTCCCTACCCCGGAGGCGCTGGAGC
AATACGACCCTGCCGCATTGAAAGCAGCCCTGGCGGATTCGCTGCAGCGTAC
GGCGCGTGATGCCGAGCTGCAGAGCTATGAAGTGCCGGCGGACTTCATTGTT
GAGACTGAGCCTTTTAGCGCTGCGAACGGTCTGCTGAGCGGTGTTGGCAAGT
TGCTGCGTCCGAATTTGAAGGATCGCTACGGTCAGCGTTTGGAGCAGATGTA
CGCGGACATCGCGGCTACGCAGGCGAACCAATTGCGTGAACTGCGCCGTGCT
GCGGCTACTCAACCGGTGATCGACACGCTGACGCAAGCTGCGGCGACCATCC
TGGGTACCGGCAGCGAGGTTGCAAGCGACGCACACTTTACTGATTTGGGCGG
TGATTCTCTGAGCGCGCTGACGTTGAGCAACTTGCTGTCTGACTTCTTTGGC
TTTGAAGTCCCGGTTGGCACGATTGTTAACCCAGCGACTAATCTGGCACAGC
TGGCGCAACATATCGAGGCGCAGCGCACGGCGGGTGACCGCCGTCCATCCTT
TACGACGGTCCACGGTGCGGATGCTACGGAAATCCGTGCAAGCGAACTGACT
CTGGACAAATTCATCGACGCTGAGACTCTGCGCGCAGCACCTGGTTTGCCGA
AGGTTACGACTGAGCCGCGTACGGTCCTGTTGAGCGGTGCCAATGGTTGGTT
GGGCCGCTTCCTGACCCTGCAGTGGCTGGAACGTTTGGCACCGGTTGGCGGT
ACCCTGATCACCATTGTGCGCGGTCGTGACGATGCAGCGGCACGTGCACGTT
TGACTCAGGCTTACGATACGGACCCAGAGCTGTCCCGCCGCTTCGCTGAGTT
GGCGGATCGCCACTTGCGTGTGGTGGCAGGTGATATCGGCGATCCGAATCTG
GGCCTGACCCCGGAGATTTGGCACCGTCTGGCAGCAGAGGTCGATCTGGTCG
TTCATCCAGCGGCCCTGGTCAACCACGTCCTGCCGTACCGCCAGCTGTTTGG
TCCGAATGTTGTTGGCACCGCCGAAGTTATCAAGTTGGCTCTGACCGAGCGC
ATCAAGCCTGTTACCTACCTGTCCACGGTTAGCGTCGCGATGGGTATTCCTG
ATTTTGAGGAGGACGGTGACATTCGTACCGTCAGCCCGGTTCGTCCGCTGGA
TGGTGGCTATGCAAATGGCTATGGCAACAGCAAGTGGGCTGGCGAGGTGCTG
CTGCGCGAGGCACATGACCTGTGTGGCCTGCCGGTTGCGACGTTTCGTAGCG
ACATGATTCTGGCCCACCCGCGCTACCGTGGCCAAGTGAATGTGCCGGACAT
GTTCACCCGTCTGCTGCTGTCCCTGCTGATCACGGGTGTGGCACCGCGTTCC
TTCTACATTGGTGATGGCGAGCGTCCGCGTGCACACTACCCGGGCCTGACCG
TCGATTTTGTTGCGGAAGCGGTTACTACCCTGGGTGCTCAGCAACGTGAGGG
TTATGTCTCGTATGACGTTATGAATCCGCACGATGACGGTATTAGCTTGGAT
GTCTTTGTGGACTGGCTGATTCGTGCGGGCCACCCAATTGACCGTGTTGACG
ACTATGATGACTGGGTGCGTCGTTTTGAAACCGCGTTGACCGCCTTGCCGGA
GAAACGTCGTGCGCAGACCGTTCTGCCGCTGCTGCATGCCTTTCGCGCGCCA
CAGGCGCCGTTGCGTGGCGCCCCTGAACCGACCGAAGTGTTTCATGCAGCGG
TGCGTACCGCTAAAGTCGGTCCGGGTGATATTCCGCACCTGGATGAAGCCCT
GATCGACAAGTACATCCGTGACCTGCGCGAGTTCGGTCTGATTTAG
SEQUENCE LISTINGS
1
5611232DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 1ttgtccatct ttatataatt
tgggggtagg gtgttcttta tgtaaaaaaa acgttttagg 60atgcatatgg cggccgcata
acttcgtata gcatacatta tacgaagtta tctagagttg 120catgcctgca
ggtccgctta ttatcactta ttcaggcgta gcaaccaggc gtttaagggc
180accaataact gccttaaaaa aattacgccc cgccctgcca ctcatcgcag
tactgttgta 240attcattaag cattctgccg acatggaagc catcacaaac
ggcatgatga acctgaatcg 300ccagcggcat cagcaccttg tcgccttgcg
tataatattt gcccatggtg aaaacggggg 360cgaagaagtt gtccatattg
gccacgttta aatcaaaact ggtgaaactc acccagggat 420tggctgagac
gaaaaacata ttctcaataa accctttagg gaaataggcc aggttttcac
480cgtaacacgc cacatcttgc gaatatatgt gtagaaactg ccggaaatcg
tcgtggtatt 540cactccagag cgatgaaaac gtttcagttt gctcatggaa
aacggtgtaa caagggtgaa 600cactatccca tatcaccagc tcaccgtctt
tcattgccat acggaattcc ggatgagcat 660tcatcaggcg ggcaagaatg
tgaataaagg ccggataaaa cttgtgctta tttttcttta 720cggtctttaa
aaaggccgta atatccagct gaacggtctg gttataggta cattgagcaa
780ctgactgaaa tgcctcaaaa tgttctttac gatgccattg ggatatatca
acggtggtat 840atccagtgat ttttttctcc attttagctt ccttagctcc
tgaaaatctc gataactcaa 900aaaatacgcc cggtagtgat cttatttcat
tatggtgaaa gttggaacct cttacgtgcc 960gatcaacgtc tcattttcgc
caaaagttgg cccagggctt cccggtatca acagggacac 1020caggatttat
ttattctgcg aagtgatctt ccgtcacagg tatttattcg actctagata
1080acttcgtata gcatacatta tacgaagtta tggatccagc ttatcgatac
cgtcaaacaa 1140atcataaaaa atttatttgc tttcaggaaa atttttctgt
ataatagatt caattgcgat 1200gacgacgaac acgcacctgc aggaggagac ca
12322232DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 2ttgtccatct
ttatataatt tgggggtagg gtgttcttta tgtaaaaaaa acgttttagg 60atgcatatgg
cggccgcata acttcgtata gcatacatta tacgaagtta tggatccagc
120ttatcgatac cgtcaaacaa atcataaaaa atttatttgc tttcaggaaa
atttttctgt 180ataatagatt caattgcgat gacgacgaac acgcacctgc
aggaggagac ca 2323340PRTAcinetobacter sp. 3Met Ser Asn His Gln Ile
Arg Ala Tyr Ala Ala Met Gln Ala Gly Glu1 5 10 15Gln Val Val Pro Tyr
Gln Phe Asp Ala Gly Glu Leu Lys Ala His Gln 20 25 30Val Glu Val Lys
Val Glu Tyr Cys Gly Leu Cys His Ser Asp Leu Ser 35 40 45Val Ile Asn
Asn Glu Trp Gln Ser Ser Val Tyr Pro Ala Val Ala Gly 50 55 60His Glu
Ile Ile Gly Thr Ile Ile Ala Leu Gly Ser Glu Ala Lys Gly65 70 75
80Leu Lys Leu Gly Gln Arg Val Gly Ile Gly Trp Thr Ala Glu Thr Cys
85 90 95Gln Ala Cys Asp Pro Cys Ile Gly Gly Asn Gln Val Leu Cys Thr
Gly 100 105 110Glu Lys Lys Ala Thr Ile Ile Gly His Ala Gly Gly Phe
Ala Asp Lys 115 120 125Val Arg Ala Gly Trp Gln Trp Val Ile Pro Leu
Pro Asp Asp Leu Asp 130 135 140Pro Glu Ser Ala Gly Pro Leu Leu Cys
Gly Gly Ile Thr Val Leu Asp145 150 155 160Pro Leu Leu Lys His Lys
Ile Gln Ala Thr His His Val Gly Val Ile 165 170 175Gly Ile Gly Gly
Leu Gly His Ile Ala Ile Lys Leu Leu Lys Ala Trp 180 185 190Gly Cys
Glu Ile Thr Ala Phe Ser Ser Asn Pro Asp Lys Thr Glu Glu 195 200
205Leu Lys Ala Asn Gly Ala Asp Gln Val Val Asn Ser Arg Asp Ala Gln
210 215 220Ala Ile Lys Gly Thr Arg Trp Lys Leu Ile Ile Leu Ser Thr
Ala Asn225 230 235 240Gly Thr Leu Asn Val Lys Ala Tyr Leu Asn Thr
Leu Ala Pro Lys Gly 245 250 255Ser Leu His Phe Leu Gly Val Thr Leu
Glu Pro Ile Pro Val Ser Val 260 265 270Gly Ala Ile Met Gly Gly Ala
Lys Ser Val Thr Ser Ser Pro Thr Gly 275 280 285Ser Pro Leu Ala Leu
Arg Gln Leu Leu Gln Phe Ala Ala Arg Lys Asn 290 295 300Ile Ala Pro
Gln Val Glu Leu Phe Pro Met Ser Gln Leu Asn Glu Ala305 310 315
320Ile Glu Arg Leu His Ser Gly Gln Ala Arg Tyr Arg Ile Val Leu Lys
325 330 335Ala Asp Phe Asp 3404314PRTAcinetobacter sp. 4Met Ala Thr
Thr Asn Val Ile His Ala Tyr Ala Ala Met Gln Ala Gly1 5 10 15Glu Ala
Leu Val Pro Tyr Ser Phe Asp Ala Gly Glu Leu Gln Pro His 20 25 30Gln
Val Glu Val Lys Val Glu Tyr Cys Gly Leu Cys His Ser Asp Val 35 40
45Ser Val Leu Asn Asn Glu Trp His Ser Ser Val Tyr Pro Val Val Ala
50 55 60Gly His Glu Val Ile Gly Thr Ile Thr Gln Leu Gly Ser Glu Ala
Lys65 70 75 80Gly Leu Lys Ile Gly Gln Arg Val Gly Ile Gly Trp Thr
Ala Glu Ser 85 90 95Cys Gln Ala Cys Asp Gln Cys Ile Ser Gly Gln Gln
Val Leu Cys Thr 100 105 110Gly Glu Asn Thr Ala Thr Ile Ile Gly His
Ala Gly Gly Phe Ala Asp 115 120 125Lys Val Arg Ala Gly Trp Gln Trp
Val Ile Pro Leu Pro Asp Glu Leu 130 135 140Asp Pro Thr Ser Ala Gly
Pro Leu Leu Cys Gly Gly Ile Thr Val Phe145 150 155 160Asp Pro Ile
Leu Lys His Gln Ile Gln Ala Ile His His Val Ala Val 165 170 175Ile
Gly Ile Gly Gly Leu Gly His Met Ala Ile Lys Leu Leu Lys Ala 180 185
190Trp Gly Cys Glu Ile Thr Ala Phe Ser Ser Asn Pro Asn Lys Thr Asp
195 200 205Glu Leu Lys Ala Met Gly Ala Asp His Val Val Asn Ser Arg
Asp Asp 210 215 220Ala Glu Ile Lys Ser Gln Gln Gly Lys Phe Asp Leu
Leu Leu Ser Thr225 230 235 240Val Asn Val Pro Leu Asn Trp Asn Ala
Tyr Leu Asn Thr Leu Ala Pro 245 250 255Asn Gly Thr Phe His Phe Leu
Gly Val Val Met Glu Pro Ile Pro Val 260 265 270Pro Val Gly Ala Leu
Leu Gly Gly Ala Lys Ser Leu Thr Ala Ser Pro 275 280 285Thr Gly Ser
Pro Ala Ala Leu Arg Lys Leu Leu Glu Phe Ala Ala Arg 290 295 300Lys
Asn Ile Ala Pro Gln Ile Glu Met Tyr305 31051020DNAEscherichia coli
5atgtcgatga taaaaagcta tgccgcaaaa gaagcgggcg gcgaactgga agtttatgag
60tacgatcccg gtgagctgag gccacaagat gttgaagtgc aggtggatta ctgcgggatc
120tgccattccg atctgtcgat gatcgataac gaatggggat tttcacaata
tccgctggtt 180gccgggcatg aggtgattgg gcgcgtggtg gcactcggga
gcgccgcgca ggataaaggt 240ttgcaggtcg gtcagcgtgt cgggattggc
tggacggcgc gtagctgtgg tcactgcgac 300gcctgtatta gcggtaatca
gatcaactgc gagcaaggtg cggtgccgac gattatgaat 360cgcggtggct
ttgccgagaa gttgcgtgcg gactggcaat gggtgattcc actgccagaa
420aatattgata tcgagtccgc cgggccgctg ttgtgcggcg gtatcacggt
ctttaaacca 480ctgttgatgc accatatcac tgctaccagc cgcgttgggg
taattggtat tggcgggctg 540gggcatatcg ctataaaact tctgcacgca
atgggatgcg aggtgacagc ctttagttct 600aatccggcga aagagcagga
agtgctggcg atgggtgccg ataaagtggt gaatagccgc 660gatccgcagg
cactgaaagc actggcgggg cagtttgatc tcattatcaa caccgtcaac
720gtcagcctcg actggcagcc ctattttgag gcgctgacct atggcggtaa
tttccatacg 780gtcggtgcgg ttctcacgcc gctgtctgtt ccggccttta
cgttaattgc gggcgatcgc 840agcgtctctg gttctgctac cggcacgcct
tatgagctgc gtaagctgat gcgttttgcc 900gcccgcagca aggttgcgcc
gaccaccgaa ctgttcccga tgtcgaaaat taacgacgcc 960atccagcatg
tgcgcgacgg taaggcgcgt taccgcgtgg tgttgaaagc cgatttttga
102061174PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 6Met Ala Val Asp Ser Pro
Asp Glu Arg Leu Gln Arg Arg Ile Ala Gln1 5 10 15Leu Phe Ala Glu Asp
Glu Gln Val Lys Ala Ala Arg Pro Leu Glu Ala 20 25 30Val Ser Ala Ala
Val Ser Ala Pro Gly Met Arg Leu Ala Gln Ile Ala 35 40 45Ala Thr Val
Met Ala Gly Tyr Ala Asp Arg Pro Ala Ala Gly Gln Arg 50 55 60Ala Phe
Glu Leu Asn Thr Asp Asp Ala Thr Gly Arg Thr Ser Leu Arg65 70 75
80Leu Leu Pro Arg Phe Glu Thr Ile Thr Tyr Arg Glu Leu Trp Gln Arg
85 90 95Val Gly Glu Val Ala Ala Ala Trp His His Asp Pro Glu Asn Pro
Leu 100 105 110Arg Ala Gly Asp Phe Val Ala Leu Leu Gly Phe Thr Ser
Ile Asp Tyr 115 120 125Ala Thr Leu Asp Leu Ala Asp Ile His Leu Gly
Ala Val Thr Val Pro 130 135 140Leu Gln Ala Ser Ala Ala Val Ser Gln
Leu Ile Ala Ile Leu Thr Glu145 150 155 160Thr Ser Pro Arg Leu Leu
Ala Ser Thr Pro Glu His Leu Asp Ala Ala 165 170 175Val Glu Cys Leu
Leu Ala Gly Thr Thr Pro Glu Arg Leu Val Val Phe 180 185 190Asp Tyr
His Pro Glu Asp Asp Asp Gln Arg Ala Ala Phe Glu Ser Ala 195 200
205Arg Arg Arg Leu Ala Asp Ala Gly Ser Leu Val Ile Val Glu Thr Leu
210 215 220Asp Ala Val Arg Ala Arg Gly Arg Asp Leu Pro Ala Ala Pro
Leu Phe225 230 235 240Val Pro Asp Thr Asp Asp Asp Pro Leu Ala Leu
Leu Ile Tyr Thr Ser 245 250 255Gly Ser Thr Gly Thr Pro Lys Gly Ala
Met Tyr Thr Asn Arg Leu Ala 260 265 270Ala Thr Met Trp Gln Gly Asn
Ser Met Leu Gln Gly Asn Ser Gln Arg 275 280 285Val Gly Ile Asn Leu
Asn Tyr Met Pro Met Ser His Ile Ala Gly Arg 290 295 300Ile Ser Leu
Phe Gly Val Leu Ala Arg Gly Gly Thr Ala Tyr Phe Ala305 310 315
320Ala Lys Ser Asp Met Ser Thr Leu Phe Glu Asp Ile Gly Leu Val Arg
325 330 335Pro Thr Glu Ile Phe Phe Val Pro Arg Val Cys Asp Met Val
Phe Gln 340 345 350Arg Tyr Gln Ser Glu Leu Asp Arg Arg Ser Val Ala
Gly Ala Asp Leu 355 360 365Asp Thr Leu Asp Arg Glu Val Lys Ala Asp
Leu Arg Gln Asn Tyr Leu 370 375 380Gly Gly Arg Phe Leu Val Ala Val
Val Gly Ser Ala Pro Leu Ala Ala385 390 395 400Glu Met Lys Thr Phe
Met Glu Ser Val Leu Asp Leu Pro Leu His Asp 405 410 415Gly Tyr Gly
Ser Thr Glu Ala Gly Ala Ser Val Leu Leu Asp Asn Gln 420 425 430Ile
Gln Arg Pro Pro Val Leu Asp Tyr Lys Leu Val Asp Val Pro Glu 435 440
445Leu Gly Tyr Phe Arg Thr Asp Arg Pro His Pro Arg Gly Glu Leu Leu
450 455 460Leu Lys Ala Glu Thr Thr Ile Pro Gly Tyr Tyr Lys Arg Pro
Glu Val465 470 475 480Thr Ala Glu Ile Phe Asp Glu Asp Gly Phe Tyr
Lys Thr Gly Asp Ile 485 490 495Val Ala Glu Leu Glu His Asp Arg Leu
Val Tyr Val Asp Arg Arg Asn 500 505 510Asn Val Leu Lys Leu Ser Gln
Gly Glu Phe Val Thr Val Ala His Leu 515 520 525Glu Ala Val Phe Ala
Ser Ser Pro Leu Ile Arg Gln Ile Phe Ile Tyr 530 535 540Gly Ser Ser
Glu Arg Ser Tyr Leu Leu Ala Val Ile Val Pro Thr Asp545 550 555
560Asp Ala Leu Arg Gly Arg Asp Thr Ala Thr Leu Lys Ser Ala Leu Ala
565 570 575Glu Ser Ile Gln Arg Ile Ala Lys Asp Ala Asn Leu Gln Pro
Tyr Glu 580 585 590Ile Pro Arg Asp Phe Leu Ile Glu Thr Glu Pro Phe
Thr Ile Ala Asn 595 600 605Gly Leu Leu Ser Gly Ile Ala Lys Leu Leu
Arg Pro Asn Leu Lys Glu 610 615 620Arg Tyr Gly Ala Gln Leu Glu Gln
Met Tyr Thr Asp Leu Ala Thr Gly625 630 635 640Gln Ala Asp Glu Leu
Leu Ala Leu Arg Arg Glu Ala Ala Asp Leu Pro 645 650 655Val Leu Glu
Thr Val Ser Arg Ala Ala Lys Ala Met Leu Gly Val Ala 660 665 670Ser
Ala Asp Met Arg Pro Asp Ala His Phe Thr Asp Leu Gly Gly Asp 675 680
685Ser Leu Ser Ala Leu Ser Phe Ser Asn Leu Leu His Glu Ile Phe Gly
690 695 700Val Glu Val Pro Val Gly Val Val Val Ser Pro Ala Asn Glu
Leu Arg705 710 715 720Asp Leu Ala Asn Tyr Ile Glu Ala Glu Arg Asn
Ser Gly Ala Lys Arg 725 730 735Pro Thr Phe Thr Ser Val His Gly Gly
Gly Ser Glu Ile Arg Ala Ala 740 745 750Asp Leu Thr Leu Asp Lys Phe
Ile Asp Ala Arg Thr Leu Ala Ala Ala 755 760 765Asp Ser Ile Pro His
Ala Pro Val Pro Ala Gln Thr Val Leu Leu Thr 770 775 780Gly Ala Asn
Gly Tyr Leu Gly Arg Phe Leu Cys Leu Glu Trp Leu Glu785 790 795
800Arg Leu Asp Lys Thr Gly Gly Thr Leu Ile Cys Val Val Arg Gly Ser
805 810 815Asp Ala Ala Ala Ala Arg Lys Arg Leu Asp Ser Ala Phe Asp
Ser Gly 820 825 830Asp Pro Gly Leu Leu Glu His Tyr Gln Gln Leu Ala
Ala Arg Thr Leu 835 840 845Glu Val Leu Ala Gly Asp Ile Gly Asp Pro
Asn Leu Gly Leu Asp Asp 850 855 860Ala Thr Trp Gln Arg Leu Ala Glu
Thr Val Asp Leu Ile Val His Pro865 870 875 880Ala Ala Leu Val Asn
His Val Leu Pro Tyr Thr Gln Leu Phe Gly Pro 885 890 895Asn Val Val
Gly Thr Ala Glu Ile Val Arg Leu Ala Ile Thr Ala Arg 900 905 910Arg
Lys Pro Val Thr Tyr Leu Ser Thr Val Gly Val Ala Asp Gln Val 915 920
925Asp Pro Ala Glu Tyr Gln Glu Asp Ser Asp Val Arg Glu Met Ser Ala
930 935 940Val Arg Val Val Arg Glu Ser Tyr Ala Asn Gly Tyr Gly Asn
Ser Lys945 950 955 960Trp Ala Gly Glu Val Leu Leu Arg Glu Ala His
Asp Leu Cys Gly Leu 965 970 975Pro Val Ala Val Phe Arg Ser Asp Met
Ile Leu Ala His Ser Arg Tyr 980 985 990Ala Gly Gln Leu Asn Val Gln
Asp Val Phe Thr Arg Leu Ile Leu Ser 995 1000 1005Leu Val Ala Thr
Gly Ile Ala Pro Tyr Ser Phe Tyr Arg Thr Asp 1010 1015 1020Ala Asp
Gly Asn Arg Gln Arg Ala His Tyr Asp Gly Leu Pro Ala 1025 1030
1035Asp Phe Thr Ala Ala Ala Ile Thr Ala Leu Gly Ile Gln Ala Thr
1040 1045 1050Glu Gly Phe Arg Thr Tyr Asp Val Leu Asn Pro Tyr Asp
Asp Gly 1055 1060 1065Ile Ser Leu Asp Glu Phe Val Asp Trp Leu Val
Glu Ser Gly His 1070 1075 1080Pro Ile Gln Arg Ile Thr Asp Tyr Ser
Asp Trp Phe His Arg Phe 1085 1090 1095Glu Thr Ala Ile Arg Ala Leu
Pro Glu Lys Gln Arg Gln Ala Ser 1100 1105 1110Val Leu Pro Leu Leu
Asp Ala Tyr Arg Asn Pro Cys Pro Ala Val 1115 1120 1125Arg Gly Ala
Ile Leu Pro Ala Lys Glu Phe Gln Ala Ala Val Gln 1130 1135 1140Thr
Ala Lys Ile Gly Pro Glu Gln Asp Ile Pro His Leu Ser Ala 1145 1150
1155Pro Leu Ile Asp Lys Tyr Val Ser Asp Leu Glu Leu Leu Gln Leu
1160 1165 1170Leu71173PRTMycobacterium smegmatis 7Met Thr Ser Asp
Val His Asp Ala Thr Asp Gly Val Thr Glu Thr Ala1 5 10 15Leu Asp Asp
Glu Gln Ser Thr Arg Arg Ile Ala Glu Leu Tyr Ala Thr 20 25 30Asp Pro
Glu Phe Ala Ala Ala Ala Pro Leu Pro Ala Val Val Asp Ala 35 40 45Ala
His Lys Pro Gly Leu Arg Leu Ala Glu Ile Leu Gln Thr Leu Phe 50 55
60Thr Gly Tyr Gly Asp Arg Pro Ala Leu Gly Tyr Arg Ala Arg Glu Leu65
70 75 80Ala Thr Asp Glu Gly Gly Arg Thr Val Thr Arg Leu Leu Pro Arg
Phe 85 90 95Asp Thr Leu Thr Tyr Ala Gln Val Trp Ser Arg Val Gln Ala
Val Ala 100 105 110Ala Ala Leu Arg His Asn Phe Ala Gln Pro Ile Tyr
Pro Gly Asp Ala 115 120 125Val Ala Thr Ile Gly Phe Ala Ser Pro Asp
Tyr Leu Thr Leu Asp Leu 130 135 140Val Cys Ala Tyr Leu Gly Leu Val
Ser Val Pro Leu Gln His Asn Ala145 150 155 160Pro Val Ser Arg Leu
Ala Pro Ile Leu Ala Glu Val Glu Pro Arg Ile
165 170 175Leu Thr Val Ser Ala Glu Tyr Leu Asp Leu Ala Val Glu Ser
Val Arg 180 185 190Asp Val Asn Ser Val Ser Gln Leu Val Val Phe Asp
His His Pro Glu 195 200 205Val Asp Asp His Arg Asp Ala Leu Ala Arg
Ala Arg Glu Gln Leu Ala 210 215 220Gly Lys Gly Ile Ala Val Thr Thr
Leu Asp Ala Ile Ala Asp Glu Gly225 230 235 240Ala Gly Leu Pro Ala
Glu Pro Ile Tyr Thr Ala Asp His Asp Gln Arg 245 250 255Leu Ala Met
Ile Leu Tyr Thr Ser Gly Ser Thr Gly Ala Pro Lys Gly 260 265 270Ala
Met Tyr Thr Glu Ala Met Val Ala Arg Leu Trp Thr Met Ser Phe 275 280
285Ile Thr Gly Asp Pro Thr Pro Val Ile Asn Val Asn Phe Met Pro Leu
290 295 300Asn His Leu Gly Gly Arg Ile Pro Ile Ser Thr Ala Val Gln
Asn Gly305 310 315 320Gly Thr Ser Tyr Phe Val Pro Glu Ser Asp Met
Ser Thr Leu Phe Glu 325 330 335Asp Leu Ala Leu Val Arg Pro Thr Glu
Leu Gly Leu Val Pro Arg Val 340 345 350Ala Asp Met Leu Tyr Gln His
His Leu Ala Thr Val Asp Arg Leu Val 355 360 365Thr Gln Gly Ala Asp
Glu Leu Thr Ala Glu Lys Gln Ala Gly Ala Glu 370 375 380Leu Arg Glu
Gln Val Leu Gly Gly Arg Val Ile Thr Gly Phe Val Ser385 390 395
400Thr Ala Pro Leu Ala Ala Glu Met Arg Ala Phe Leu Asp Ile Thr Leu
405 410 415Gly Ala His Ile Val Asp Gly Tyr Gly Leu Thr Glu Thr Gly
Ala Val 420 425 430Thr Arg Asp Gly Val Ile Val Arg Pro Pro Val Ile
Asp Tyr Lys Leu 435 440 445Ile Asp Val Pro Glu Leu Gly Tyr Phe Ser
Thr Asp Lys Pro Tyr Pro 450 455 460Arg Gly Glu Leu Leu Val Arg Ser
Gln Thr Leu Thr Pro Gly Tyr Tyr465 470 475 480Lys Arg Pro Glu Val
Thr Ala Ser Val Phe Asp Arg Asp Gly Tyr Tyr 485 490 495His Thr Gly
Asp Val Met Ala Glu Thr Ala Pro Asp His Leu Val Tyr 500 505 510Val
Asp Arg Arg Asn Asn Val Leu Lys Leu Ala Gln Gly Glu Phe Val 515 520
525Ala Val Ala Asn Leu Glu Ala Val Phe Ser Gly Ala Ala Leu Val Arg
530 535 540Gln Ile Phe Val Tyr Gly Asn Ser Glu Arg Ser Phe Leu Leu
Ala Val545 550 555 560Val Val Pro Thr Pro Glu Ala Leu Glu Gln Tyr
Asp Pro Ala Ala Leu 565 570 575Lys Ala Ala Leu Ala Asp Ser Leu Gln
Arg Thr Ala Arg Asp Ala Glu 580 585 590Leu Gln Ser Tyr Glu Val Pro
Ala Asp Phe Ile Val Glu Thr Glu Pro 595 600 605Phe Ser Ala Ala Asn
Gly Leu Leu Ser Gly Val Gly Lys Leu Leu Arg 610 615 620Pro Asn Leu
Lys Asp Arg Tyr Gly Gln Arg Leu Glu Gln Met Tyr Ala625 630 635
640Asp Ile Ala Ala Thr Gln Ala Asn Gln Leu Arg Glu Leu Arg Arg Ala
645 650 655Ala Ala Thr Gln Pro Val Ile Asp Thr Leu Thr Gln Ala Ala
Ala Thr 660 665 670Ile Leu Gly Thr Gly Ser Glu Val Ala Ser Asp Ala
His Phe Thr Asp 675 680 685Leu Gly Gly Asp Ser Leu Ser Ala Leu Thr
Leu Ser Asn Leu Leu Ser 690 695 700Asp Phe Phe Gly Phe Glu Val Pro
Val Gly Thr Ile Val Asn Pro Ala705 710 715 720Thr Asn Leu Ala Gln
Leu Ala Gln His Ile Glu Ala Gln Arg Thr Ala 725 730 735Gly Asp Arg
Arg Pro Ser Phe Thr Thr Val His Gly Ala Asp Ala Thr 740 745 750Glu
Ile Arg Ala Ser Glu Leu Thr Leu Asp Lys Phe Ile Asp Ala Glu 755 760
765Thr Leu Arg Ala Ala Pro Gly Leu Pro Lys Val Thr Thr Glu Pro Arg
770 775 780Thr Val Leu Leu Ser Gly Ala Asn Gly Trp Leu Gly Arg Phe
Leu Thr785 790 795 800Leu Gln Trp Leu Glu Arg Leu Ala Pro Val Gly
Gly Thr Leu Ile Thr 805 810 815Ile Val Arg Gly Arg Asp Asp Ala Ala
Ala Arg Ala Arg Leu Thr Gln 820 825 830Ala Tyr Asp Thr Asp Pro Glu
Leu Ser Arg Arg Phe Ala Glu Leu Ala 835 840 845Asp Arg His Leu Arg
Val Val Ala Gly Asp Ile Gly Asp Pro Asn Leu 850 855 860Gly Leu Thr
Pro Glu Ile Trp His Arg Leu Ala Ala Glu Val Asp Leu865 870 875
880Val Val His Pro Ala Ala Leu Val Asn His Val Leu Pro Tyr Arg Gln
885 890 895Leu Phe Gly Pro Asn Val Val Gly Thr Ala Glu Val Ile Lys
Leu Ala 900 905 910Leu Thr Glu Arg Ile Lys Pro Val Thr Tyr Leu Ser
Thr Val Ser Val 915 920 925Ala Met Gly Ile Pro Asp Phe Glu Glu Asp
Gly Asp Ile Arg Thr Val 930 935 940Ser Pro Val Arg Pro Leu Asp Gly
Gly Tyr Ala Asn Gly Tyr Gly Asn945 950 955 960Ser Lys Trp Ala Gly
Glu Val Leu Leu Arg Glu Ala His Asp Leu Cys 965 970 975Gly Leu Pro
Val Ala Thr Phe Arg Ser Asp Met Ile Leu Ala His Pro 980 985 990Arg
Tyr Arg Gly Gln Val Asn Val Pro Asp Met Phe Thr Arg Leu Leu 995
1000 1005Leu Ser Leu Leu Ile Thr Gly Val Ala Pro Arg Ser Phe Tyr
Ile 1010 1015 1020Gly Asp Gly Glu Arg Pro Arg Ala His Tyr Pro Gly
Leu Thr Val 1025 1030 1035Asp Phe Val Ala Glu Ala Val Thr Thr Leu
Gly Ala Gln Gln Arg 1040 1045 1050Glu Gly Tyr Val Ser Tyr Asp Val
Met Asn Pro His Asp Asp Gly 1055 1060 1065Ile Ser Leu Asp Val Phe
Val Asp Trp Leu Ile Arg Ala Gly His 1070 1075 1080Pro Ile Asp Arg
Val Asp Asp Tyr Asp Asp Trp Val Arg Arg Phe 1085 1090 1095Glu Thr
Ala Leu Thr Ala Leu Pro Glu Lys Arg Arg Ala Gln Thr 1100 1105
1110Val Leu Pro Leu Leu His Ala Phe Arg Ala Pro Gln Ala Pro Leu
1115 1120 1125Arg Gly Ala Pro Glu Pro Thr Glu Val Phe His Ala Ala
Val Arg 1130 1135 1140Thr Ala Lys Val Gly Pro Gly Asp Ile Pro His
Leu Asp Glu Ala 1145 1150 1155Leu Ile Asp Lys Tyr Ile Arg Asp Leu
Arg Glu Phe Gly Leu Ile 1160 1165 11708209PRTEscherichia coli 8Met
Val Asp Met Lys Thr Thr His Thr Ser Leu Pro Phe Ala Gly His1 5 10
15Thr Leu His Phe Val Glu Phe Asp Pro Ala Asn Phe Cys Glu Gln Asp
20 25 30Leu Leu Trp Leu Pro His Tyr Ala Gln Leu Gln His Ala Gly Arg
Lys 35 40 45Arg Lys Thr Glu His Leu Ala Gly Arg Ile Ala Ala Val Tyr
Ala Leu 50 55 60Arg Glu Tyr Gly Tyr Lys Cys Val Pro Ala Ile Gly Glu
Leu Arg Gln65 70 75 80Pro Val Trp Pro Ala Glu Val Tyr Gly Ser Ile
Ser His Cys Gly Thr 85 90 95Thr Ala Leu Ala Val Val Ser Arg Gln Pro
Ile Gly Ile Asp Ile Glu 100 105 110Glu Ile Phe Ser Val Gln Thr Ala
Arg Glu Leu Thr Asp Asn Ile Ile 115 120 125Thr Pro Ala Glu His Glu
Arg Leu Ala Asp Cys Gly Leu Ala Phe Ser 130 135 140Leu Ala Leu Thr
Leu Ala Phe Ser Ala Lys Glu Ser Ala Phe Lys Ala145 150 155 160Ser
Glu Ile Gln Thr Asp Ala Gly Phe Leu Asp Tyr Gln Ile Ile Ser 165 170
175Trp Asn Lys Gln Gln Val Ile Ile His Arg Glu Asn Glu Met Phe Ala
180 185 190Val His Trp Gln Ile Lys Glu Lys Ile Val Ile Thr Leu Cys
Gln His 195 200 205Asp970DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 9aaaaacagca acaatgtgag ctttgttgta attatattgt
aaacatattg attccgggga 60tccgtcgacc 701068DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 10aaacggagcc tttcggctcc gttattcatt tacgcggctt
caactttcct gtaggctgga 60gctgcttc 681123DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 11cgggcaggtg ctatgaccag gac 231223DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 12cgcggcgttg accggcagcc tgg 23135659DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 13atgatcatca aacctaaaat tcgtggattt atctgtacaa
caacgcaccc agtgggttgt 60gaagcgaacg taaaagaaca aattgcctac acaaaagcac
aaggtccgat caaaaacgca 120cctaagcgcg tgttggttgt cggatcgtct
agcggctatg gtctgtcatc acgcatcgct 180gcggcgtttg gcggtggtgc
ggcgacgatc ggcgtatttt tcgaaaagcc gggcactgac 240aaaaaaccag
gtactgcggg tttctacaat gcagcagcgt ttgacaagct agcgcatgaa
300gcgggcttgt acgcaaaaag cctgaacggc gatgcgttct cgaacgaagc
gaagcaaaaa 360gcgattgagc tgattaagca agacctcggc cagattgatt
tggtggttta ctcgttggct 420tctccagtgc gtaagatgcc agacacgggt
gagctagtgc gctctgcact aaaaccgatc 480ggcgaaacgt acacctctac
cgcggtagat accaataaag atgtgatcat tgaagccagt 540gttgaacctg
cgaccgagca agaaatcgct gacactgtca ccgtgatggg cggtcaagat
600tgggaactgt ggatccaagc actggaagag gcgggtgttc ttgctgaagg
ttgcaaaacc 660gtggcgtaca gctacatcgg tactgaattg acttggccaa
tttactggga tggcgcttta 720ggccgtgcca agatggacct agatcgcgca
gcgacagcgc tgaacgaaaa gctggcagcg 780aaaggtggta ccgcgaacgt
tgcagttttg aaatcagtgg tgactcaagc aagctctgcg 840attcctgtga
tgccgctcta catcgcaatg gtgttcaaga agatgcgtga acagggcgtg
900catgaaggct gtatggagca gatctaccgc atgttcagtc aacgtctgta
caaagaagat 960ggttcagcgc cggaagtgga tgatcacaat cgtctgcgtt
tggatgactg ggaactgcgt 1020gatgacattc agcagcactg ccgtgatctg
tggccacaaa tcaccacaga gaacctgcgt 1080gagctgaccg attacgacat
gtacaaagaa gagttcatca agctgtttgg ctttggcatt 1140gaaggcattg
attacgatgc tgacgtcaat ccagaagtcg aatttgatgt gattgatatc
1200gagtaattta gtgactgagc gtacatgtat acgaagatta ttggtactgg
cagctatctg 1260cccgaacaag tgcggactaa cgccgatctg gaaaaaatgg
ttgagacctc tgacgagtgg 1320attgtcactc gtacaggtat tcgtaaacgc
catatcgccg cgccgaatga aactgtcgcg 1380acgatgggct ttaccgctgc
gaatcgcgcg attgagatgg cggggatcga taaagaccaa 1440attggcttga
ttgtggtggc taccacatca gcaacgcatg catttccaag cgcggcatgt
1500cagattcaaa gtatgctcgg tattaaaggt tgcccggcgt ttgatgtcgc
ggcagcgtgc 1560gcaggtttca cctacgcgtt aagcatcgcc gaccagtacg
ttaaatccgg cgcggttaaa 1620cacgcgctgg tggtcggttc cgatgtatta
gcccgcactt gcgatcctgg cgatcgcggt 1680acgatcatta ttttcggcga
tggcgcaggc gcggccgtac tgagcgcttc tgaagaaccg 1740ggtattatct
ccactcatct tcatgccgat ggccgttacg gtgaattact gaccctgccg
1800aatgccgatc gcgtaaatcc ggataacccg atttacctga caatggcggg
caatgaagtc 1860tttaaagtgg cggtcactga actggcgcat attgtcgatg
agacgctggc ggctaataac 1920ctggatcgct cagaactcga ttggctggtg
ccgcatcagg ctaacctgcg tatcattagc 1980gcgacagcga aaaaactcgg
catgtcgatg gacaatgtcg tcgtcacgct ggacaggcac 2040ggcaatacct
ccgcggcttc tgtgccgtgc gcgctggatg aagccgtgcg tgacgggcga
2100attaaagccg gtcagctggt attgcttgaa gccttcgggg gtggattcac
ctggggctcc 2160gcgctgattc gtttctagta taaggattta aacatgacgc
aatttgcatt tgtgttcccc 2220ggtcagggtt ctcagagcgt tgggatgttg
gccgagatgg cggcaaatta ccctatcgta 2280gaagaaacgt ttgctgaagc
ttctgcggct ctgggatatg atctgtgggc gctcacccag 2340caaggtccag
cggaagaact gaataaaacc tggcagacgc agccggcgtt attaaccgct
2400tccgtcgcgc tttggcgcgt ttggcagcag cagggcggta aaatgcctgc
gttaatggca 2460ggtcacagcc tgggcgaata ttccgcgctg gtttgcgctg
gcgtcatcaa ctttgctgat 2520gccgttcgtc tggtggaaat gcgcggtaaa
ttcatgcagg aagcggttcc ggaaggcact 2580ggcggcatgt ctgcgatcat
cgggctggat gatgcctcta ttgctaaagc ctgtgaagaa 2640tctgccgaag
ggcaggttgt ttcgccggtt aactttaact cgccgggaca ggtggttatc
2700gccgggcata aagaggcggt agaacgtgcg ggcgcagcct gtaaagccgc
tggcgcgaaa 2760cgcgcgctgc cgctgccggt gagcgtaccg tcgcactgcg
cgctgatgaa accagcggca 2820gataagctgg cggttgaatt agccaaaatt
acctttagcg cgccaacggt gccggtagtg 2880aacaacgttg acgtgaaatg
tgaaaccgat gccgccgcta tccgcgatgc gctggttcgc 2940cagttgtaca
atccggtaca gtggacgaag agcgtggaat ttatcgcggc gcagggcgtt
3000gaacatcttt atgaagtggg tccaggtaaa gtcctcactg gtctgacgaa
acgtattgtc 3060gacaccctga cagcgtcggc gctgaacgag ccggcggcgc
tgtctgcggc acttacgcaa 3120taaaagagga aaaccatgag ctttgaagga
aagattgcgc tggtgactgg tgcaagccgt 3180ggcataggcc gcgcaattgc
agagactctc gttgcccgcg gcgcgaaagt tatcgggact 3240gcgaccagtg
aaaatggtgc gaagaacatt agcgactatt taggtgctaa cgggaaaggt
3300ttgatgttga atgtgaccga tcctgcatct attgaatctg ttctggaaaa
tattcgcgca 3360gaatttggtg aagtggatat cctggttaat aatgccggta
tcactcgtga taatctgttg 3420atgcgaatga aagatgatga gtggaacgat
attatcgaaa ccaacttatc atccgttttc 3480cgcctgtcaa aagcggtaat
gcgcgctatg atgaaaaagc gttgtggtcg cattatcact 3540attggttctg
tggttggtac catgggaaat gcaggtcagg caaactacgc tgcggcgaaa
3600gcgggcctga tcggtttcag taaatcactg gcgcgtgaag ttgcgtcccg
tggtattact 3660gtcaatgttg tggctccggg ttttattgaa acggacatga
cgcgtgcgct gtctgacgat 3720cagcgtgcgg gtatcctggc gcaggtgcct
gcgggtcgcc tcggcggcgc tcaggaaatc 3780gccagtgcgg ttgcattttt
agcctctgac gaagcgagtt acatcactgg tgagactctg 3840cacgtcaacg
gcggaatgta catggtttaa ttttaaggtt tacataaaac atggtagata
3900aacgcgaatc ctatacaaaa gaagaccttc ttgcctctgg tcgtggtgaa
ctgtttggcg 3960ctaaagggcc gcaactccct gcaccgaaca tgctgatgat
ggaccgcgtc gttaagatga 4020ccgaaacggg cggcaatttc gacaaaggct
atgtcgaagc cgagctggat atcaatccgg 4080atctatggtt cttcggatgc
cactttatcg gcgatccggt gatgcccggt tgtctgggtc 4140tggatgctat
gtggcaattg gtgggattct acctgggctg gttgggcggc gaaggcaaag
4200gccgcgctct gggcgtgggc gaagtgaaat ttaccggcca ggttctgccg
acagccagga 4260aagtcaccta tcgtattcat ttcaaacgta tcgtaaaccg
tcgcctgatc atgggcctgg 4320cggacggtga ggttctggtg gatggtcgcc
tgatctatac cgcacacgat ttgaaagtcg 4380gtttgttcca ggatacttcc
gcgttctaaa aggaggcaac aaaatgaatc gccgcgttgt 4440cattacgggt
attggtgcag tgacgccggt gggtaacaac gctgatagct tctggtgcag
4500catcaaagag ggtaaatgtg gcattgacaa gatcaaagcg tttgacgcaa
ccgatttcaa 4560agttaagctg gctgccgaag tgaaggactt caccccggag
gactttatcg acaagcgtga 4620ggcgaaccgt atggaccgtt ttagccagtt
tgcgatcgtt gcggcggatg aggcaatcaa 4680ggacagcaaa ctggacctgg
agtcgattga taagaatcgt ttcggcgtca ttgttggtag 4740cggcattggc
ggcatcggca ccattgagaa gcaggatgaa aagctgatta ccaaaggtcc
4800gggtcgtgtg agccctatga ctattccgat gatcattgcg aatatggcaa
gcggtaatct 4860ggcgattcgt tatggcgcta aaggtatttg cacgaccatt
gtcaccgcat gtgcgagcgc 4920gaacaacagc attggtgagt ccttccgtaa
cattaagttt ggttatagcg acgttatgat 4980ctctggtggt agcgaagcag
gtatcacccc gttgagcctg gcgggttttg cctcgatgaa 5040ggccgtgacc
aaatctgagg acccgaagcg cgccagcatc ccgttcgata aggatcgcag
5100cggttttgtg atgggcgagg gcagcggtat cgttatcttg gaagagttgg
agcacgcgct 5160gaagcgtggt gccaaaatct atgccgagat cgttggctat
ggtgcgacct gcgacgcata 5220tcatatcacg agcccagcgc cgaatggtga
aggtggtgca cgtgcaatga aactggcaat 5280ggaagaagat aatgtccgcc
cagaggacat ttcctatatc aacgcgcacg gtacgagcac 5340ggcgtacaat
gacagcttcg aaacccaagc gatcaagacg gtcctgggtg aatacgccta
5400caaagtgccg gtgtctagca ccaagagcat gaccggccac ctgctgggcg
ctggcggtgc 5460agtcgaagcg attatctgtg ccaaagctat tgaagagggt
ttcattccgc cgaccatcgg 5520ctacaaagag gcggatccgg aatgcgacct
ggattacgtt cctaacgagg gccgtaatgc 5580agaagtcaac tacgttctgt
ccaacagcct gggcttcggt ggccataatg cgactctgct 5640gttcaaaaag
tacaaatga 56591440DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic oligonucleotide" 14gcagttattg
gtgcccttaa acgcctggtt gctacgcctg 401527DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 15gagccaatat gcgagaacac ccgagaa
271648DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 16cgctgaacgt attgcaggcc
gagttgctgc accgctcccg ccaggcag 481751DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 17ggaattgcca cggtgcggca ggctccatac gcgaggccag
gttatccaac g 511835DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic oligonucleotide" 18aatcaccagc
actaaagtgc gcggttcgtt acccg 351934DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 19atctgccgtg gattgcagag tctattcagc tacg
342030DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 20gcaattccat atgacgagcg atgttcacga
302129DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 21ccgctcgagt aaatcagacc gaactcgcg
292260DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 22atgggcagca gccatcatca
tcatcatcac agcagcggcc tggtgccgcg cggcagccat 602343DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 23acggatcccc ggaatgcgca acgcaattaa tgtaagttag cgc
432446DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 24tgcgtcatcg ccattgaatt
cctaaatcag accgaactcg cgcagg 462520DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 25attccgggga tccgtcgacc 202623DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide" 26aatggcgatg acgcatcctc acg 232739DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 27acggaaagga gctagcacat gggcagcagc catcatcat
392827DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 28gtaaaggatg gacggcggtc acccgcc
272925DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 29cacggcgggt gaccgccgtc catcc
253043DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 30ttaattccgg ggatccctaa atcagaccga
actcgcgcag gtc 433124DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic primer" 31cagccgttta ttgccgactg
gatg 243227DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 32ctgttttatc agaccgcttc
tgcgttc 273320DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 33gcactcgacc ggaattatcg
203425DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 34gcactacgcg tactgtgagc cagag
253545DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 35gaggaataaa ccatgacgag cgatgttcac
gacgcgaccg acggc 453625DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 36ctaaatcaga ccgaactcgc gcagg 253731DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 37catggtttat tcctccttat ttaatcgata c 313828DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 38tgacctgcgc gagttcggtc tgatttag 28391168PRTMycobacterium
smegmatis 39Met Thr Ile Glu Thr Arg Glu Asp Arg Phe Asn Arg Arg Ile
Asp His1 5 10 15Leu Phe Glu Thr Asp Pro Gln Phe Ala Ala Ala Arg Pro
Asp Glu Ala 20 25 30Ile Ser Ala Ala Ala Ala Asp Pro Glu Leu Arg Leu
Pro Ala Ala Val 35 40 45Lys Gln Ile Leu Ala Gly Tyr Ala Asp Arg Pro
Ala Leu Gly Lys Arg 50 55 60Ala Val Glu Phe Val Thr Asp Glu Glu Gly
Arg Thr Thr Ala Lys Leu65 70 75 80Leu Pro Arg Phe Asp Thr Ile Thr
Tyr Arg Gln Leu Ala Gly Arg Ile 85 90 95Gln Ala Val Thr Asn Ala Trp
His Asn His Pro Val Asn Ala Gly Asp 100 105 110Arg Val Ala Ile Leu
Gly Phe Thr Ser Val Asp Tyr Thr Thr Ile Asp 115 120 125Ile Ala Leu
Leu Glu Leu Gly Ala Val Ser Val Pro Leu Gln Thr Ser 130 135 140Ala
Pro Val Ala Gln Leu Gln Pro Ile Val Ala Glu Thr Glu Pro Lys145 150
155 160Val Ile Ala Ser Ser Val Asp Phe Leu Ala Asp Ala Val Ala Leu
Val 165 170 175Glu Ser Gly Pro Ala Pro Ser Arg Leu Val Val Phe Asp
Tyr Ser His 180 185 190Glu Val Asp Asp Gln Arg Glu Ala Phe Glu Ala
Ala Lys Gly Lys Leu 195 200 205Ala Gly Thr Gly Val Val Val Glu Thr
Ile Thr Asp Ala Leu Asp Arg 210 215 220Gly Arg Ser Leu Ala Asp Ala
Pro Leu Tyr Val Pro Asp Glu Ala Asp225 230 235 240Pro Leu Thr Leu
Leu Ile Tyr Thr Ser Gly Ser Thr Gly Thr Pro Lys 245 250 255Gly Ala
Met Tyr Pro Glu Ser Lys Thr Ala Thr Met Trp Gln Ala Gly 260 265
270Ser Lys Ala Arg Trp Asp Glu Thr Leu Gly Val Met Pro Ser Ile Thr
275 280 285Leu Asn Phe Met Pro Met Ser His Val Met Gly Arg Gly Ile
Leu Cys 290 295 300Ser Thr Leu Ala Ser Gly Gly Thr Ala Tyr Phe Ala
Ala Arg Ser Asp305 310 315 320Leu Ser Thr Phe Leu Glu Asp Leu Ala
Leu Val Arg Pro Thr Gln Leu 325 330 335Asn Phe Val Pro Arg Ile Trp
Asp Met Leu Phe Gln Glu Tyr Gln Ser 340 345 350Arg Leu Asp Asn Arg
Arg Ala Glu Gly Ser Glu Asp Arg Ala Glu Ala 355 360 365Ala Val Leu
Glu Glu Val Arg Thr Gln Leu Leu Gly Gly Arg Phe Val 370 375 380Ser
Ala Leu Thr Gly Ser Ala Pro Ile Ser Ala Glu Met Lys Ser Trp385 390
395 400Val Glu Asp Leu Leu Asp Met His Leu Leu Glu Gly Tyr Gly Ser
Thr 405 410 415Glu Ala Gly Ala Val Phe Ile Asp Gly Gln Ile Gln Arg
Pro Pro Val 420 425 430Ile Asp Tyr Lys Leu Val Asp Val Pro Asp Leu
Gly Tyr Phe Ala Thr 435 440 445Asp Arg Pro Tyr Pro Arg Gly Glu Leu
Leu Val Lys Ser Glu Gln Met 450 455 460Phe Pro Gly Tyr Tyr Lys Arg
Pro Glu Ile Thr Ala Glu Met Phe Asp465 470 475 480Glu Asp Gly Tyr
Tyr Arg Thr Gly Asp Ile Val Ala Glu Leu Gly Pro 485 490 495Asp His
Leu Glu Tyr Leu Asp Arg Arg Asn Asn Val Leu Lys Leu Ser 500 505
510Gln Gly Glu Phe Val Thr Val Ser Lys Leu Glu Ala Val Phe Gly Asp
515 520 525Ser Pro Leu Val Arg Gln Ile Tyr Val Tyr Gly Asn Ser Ala
Arg Ser 530 535 540Tyr Leu Leu Ala Val Val Val Pro Thr Glu Glu Ala
Leu Ser Arg Trp545 550 555 560Asp Gly Asp Glu Leu Lys Ser Arg Ile
Ser Asp Ser Leu Gln Asp Ala 565 570 575Ala Arg Ala Ala Gly Leu Gln
Ser Tyr Glu Ile Pro Arg Asp Phe Leu 580 585 590Val Glu Thr Thr Pro
Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile 595 600 605Arg Lys Leu
Ala Arg Pro Lys Leu Lys Ala His Tyr Gly Glu Arg Leu 610 615 620Glu
Gln Leu Tyr Thr Asp Leu Ala Glu Gly Gln Ala Asn Glu Leu Arg625 630
635 640Glu Leu Arg Arg Asn Gly Ala Asp Arg Pro Val Val Glu Thr Val
Ser 645 650 655Arg Ala Ala Val Ala Leu Leu Gly Ala Ser Val Thr Asp
Leu Arg Ser 660 665 670Asp Ala His Phe Thr Asp Leu Gly Gly Asp Ser
Leu Ser Ala Leu Ser 675 680 685Phe Ser Asn Leu Leu His Glu Ile Phe
Asp Val Asp Val Pro Val Gly 690 695 700Val Ile Val Ser Pro Ala Thr
Asp Leu Ala Gly Val Ala Ala Tyr Ile705 710 715 720Glu Gly Glu Leu
Arg Gly Ser Lys Arg Pro Thr Tyr Ala Ser Val His 725 730 735Gly Arg
Asp Ala Thr Glu Val Arg Ala Arg Asp Leu Ala Leu Gly Lys 740 745
750Phe Ile Asp Ala Lys Thr Leu Ser Ala Ala Pro Gly Leu Pro Arg Ser
755 760 765Gly Thr Glu Ile Arg Thr Val Leu Leu Thr Gly Ala Thr Gly
Phe Leu 770 775 780Gly Arg Tyr Leu Ala Leu Glu Trp Leu Glu Arg Met
Asp Leu Val Asp785 790 795 800Gly Lys Val Ile Cys Leu Val Arg Ala
Arg Ser Asp Asp Glu Ala Arg 805 810 815Ala Arg Leu Asp Ala Thr Phe
Asp Thr Gly Asp Ala Thr Leu Leu Glu 820 825 830His Tyr Arg Ala Leu
Ala Ala Asp His Leu Glu Val Ile Ala Gly Asp 835 840 845Lys Gly Glu
Ala Asp Leu Gly Leu Asp His Asp Thr Trp Gln Arg Leu 850 855 860Ala
Asp Thr Val Asp Leu Ile Val Asp Pro Ala Ala Leu Val Asn His865 870
875 880Val Leu Pro Tyr Ser Gln Met Phe Gly Pro Asn Ala Leu Gly Thr
Ala 885 890 895Glu Leu Ile Arg Ile Ala Leu Thr Thr Thr Ile Lys Pro
Tyr Val Tyr 900 905 910Val Ser Thr Ile Gly Val Gly Gln Gly Ile Ser
Pro Glu Ala Phe Val 915 920 925Glu Asp Ala Asp Ile Arg Glu Ile Ser
Ala Thr Arg Arg Val Asp Asp 930 935 940Ser Tyr Ala Asn Gly Tyr Gly
Asn Ser Lys Trp Ala Gly Glu Val Leu945 950 955 960Leu Arg Glu Ala
His Asp Trp Cys Gly Leu Pro Val Ser Val Phe Arg 965 970 975Cys Asp
Met Ile Leu Ala Asp Thr Thr Tyr Ser Gly Gln Leu Asn Leu 980 985
990Pro Asp Met Phe Thr Arg Leu Met Leu Ser Leu Val Ala Thr Gly Ile
995 1000 1005Ala Pro Gly Ser Phe Tyr Glu Leu Asp Ala Asp Gly Asn
Arg Gln 1010 1015 1020Arg Ala His Tyr Asp Gly Leu Pro Val Glu Phe
Ile Ala Glu Ala 1025 1030 1035Ile Ser Thr Ile Gly Ser Gln Val Thr
Asp Gly Phe Glu Thr Phe 1040 1045 1050His Val Met Asn Pro Tyr Asp
Asp Gly Ile Gly Leu Asp Glu Tyr 1055 1060 1065Val Asp Trp Leu Ile
Glu Ala Gly Tyr Pro Val His Arg Val Asp 1070 1075 1080Asp Tyr Ala
Thr Trp Leu Ser Arg Phe Glu Thr Ala Leu Arg Ala 1085 1090 1095Leu
Pro Glu Arg Gln Arg Gln Ala Ser Leu Leu Pro Leu Leu His 1100 1105
1110Asn Tyr Gln Gln Pro Ser Pro Pro Val Cys Gly Ala Met Ala Pro
1115 1120 1125Thr Asp Arg Phe Arg Ala Ala Val Gln Asp Ala Lys Ile
Gly Pro 1130 1135 1140Asp Lys Asp Ile Pro His Val Thr Ala Asp Val
Ile Val Lys Tyr 1145 1150 1155Ile Ser Asn Leu Gln Met Leu Gly Leu
Leu 1160 1165401168PRTMycobacterium tuberculosis 40Met Ser Ile Asn
Asp Gln Arg Leu Thr Arg Arg Val Glu Asp Leu Tyr1 5 10 15Ala Ser Asp
Ala Gln Phe Ala Ala Ala Ser Pro Asn Glu Ala Ile Thr 20 25 30Gln Ala
Ile Asp Gln Pro Gly Val Ala Leu Pro Gln Leu Ile Arg Met 35 40 45Val
Met Glu Gly Tyr Ala Asp Arg Pro Ala Leu Gly Gln Arg Ala Leu 50 55
60Arg Phe Val Thr Asp Pro Asp Ser Gly Arg Thr Met Val Glu Leu Leu65
70 75 80Pro Arg Phe Glu Thr Ile Thr Tyr Arg Glu Leu Trp Ala Arg Ala
Gly 85 90 95Thr Leu Ala Thr Ala Leu Ser Ala Glu Pro Ala Ile Arg Pro
Gly Asp 100 105 110Arg Val Cys Val Leu Gly Phe Asn Ser Val Asp Tyr
Thr Thr Ile Asp 115 120 125Ile Ala Leu Ile Arg Leu Gly Ala Val Ser
Val Pro Leu Gln Thr Ser 130 135 140Ala Pro Val Thr Gly Leu Arg Pro
Ile Val Thr Glu Thr Glu Pro Thr145 150 155 160Met Ile Ala Thr Ser
Ile Asp Asn Leu Gly Asp Ala Val Glu Val Leu 165 170 175Ala Gly His
Ala Pro Ala Arg Leu Val Val Phe Asp Tyr His Gly Lys 180 185 190Val
Asp Thr His Arg Glu Ala Val Glu Ala Ala Arg Ala Arg Leu Ala 195 200
205Gly Ser Val Thr Ile Asp Thr Leu Ala Glu Leu Ile Glu Arg Gly Arg
210 215 220Ala Leu Pro Ala Thr Pro Ile Ala Asp Ser Ala Asp Asp Ala
Leu Ala225 230 235 240Leu Leu Ile Tyr Thr Ser Gly Ser Thr Gly Ala
Pro Lys Gly Ala Met 245 250 255Tyr Arg Glu Ser Gln Val Met Ser Phe
Trp Arg Lys Ser Ser Gly Trp 260 265 270Phe Glu Pro Ser Gly Tyr Pro
Ser Ile Thr Leu Asn Phe Met Pro Met 275 280 285Ser His Val Gly Gly
Arg Gln Val Leu Tyr Gly Thr Leu Ser Asn Gly 290 295 300Gly Thr Ala
Tyr Phe Val Ala Lys Ser Asp Leu Ser Thr Leu Phe Glu305 310 315
320Asp Leu Ala Leu Val Arg Pro Thr Glu Leu Cys Phe Val Pro Arg Ile
325 330 335Trp Asp Met Val Phe Ala Glu Phe His Ser Glu Val Asp Arg
Arg Leu 340 345 350Val Asp Gly Ala Asp Arg Ala Ala Leu Glu Ala Gln
Val Lys Ala Glu 355 360 365Leu Arg Glu Asn Val Leu Gly Gly Arg Phe
Val Met Ala Leu Thr Gly 370 375 380Ser Ala Pro Ile Ser Ala Glu Met
Thr Ala Trp Val Glu Ser Leu Leu385 390 395 400Ala Asp Val His Leu
Val Glu Gly Tyr Gly Ser Thr Glu Ala Gly Met 405 410 415Val Leu Asn
Asp Gly Met Val Arg Arg Pro Ala Val Ile Asp Tyr Lys 420 425 430Leu
Val Asp Val Pro Glu Leu Gly Tyr Phe Gly Thr Asp Gln Pro Tyr 435 440
445Pro Arg Gly Glu Leu Leu Val Lys Thr Gln Thr Met Phe Pro Gly Tyr
450 455 460Tyr Gln Arg Pro Asp Val Thr Ala Glu Val Phe Asp Pro Asp
Gly Phe465 470 475 480Tyr Arg Thr Gly Asp Ile Met Ala Lys Val Gly
Pro Asp Gln Phe Val 485 490 495Tyr Leu Asp Arg Arg Asn Asn Val Leu
Lys Leu Ser Gln Gly Glu Phe 500 505 510Ile Ala Val Ser Lys Leu Glu
Ala Val Phe Gly Asp Ser Pro Leu Val 515 520 525Arg Gln Ile Phe Ile
Tyr Gly Asn Ser Ala Arg Ala Tyr Pro Leu Ala 530 535 540Val Val Val
Pro Ser Gly Asp Ala Leu Ser Arg His Gly Ile Glu Asn545 550 555
560Leu Lys Pro Val Ile Ser Glu Ser Leu Gln Glu Val Ala Arg Ala Ala
565 570 575Gly Leu Gln Ser Tyr Glu Ile Pro Arg Asp Phe Ile Ile Glu
Thr Thr 580 585 590Pro Phe Thr Leu Glu Asn Gly Leu Leu Thr Gly Ile
Arg Lys Leu Ala 595 600 605Arg Pro Gln Leu Lys Lys Phe Tyr Gly Glu
Arg Leu Glu Arg Leu Tyr 610 615 620Thr Glu Leu Ala Asp Ser Gln Ser
Asn Glu Leu Arg Glu Leu Arg Gln625 630 635 640Ser Gly Pro Asp Ala
Pro Val Leu Pro Thr Leu Cys Arg Ala Ala Ala 645 650 655Ala Leu Leu
Gly Ser Thr Ala Ala Asp Val Arg Pro Asp Ala His Phe 660 665 670Ala
Asp Leu Gly Gly Asp Ser Leu Ser Ala Leu Ser Leu Ala Asn Leu 675 680
685Leu His Glu Ile Phe Gly Val Asp Val Pro Val Gly Val Ile Val Ser
690 695 700Pro Ala Ser Asp Leu Arg Ala Leu Ala Asp His Ile Glu Ala
Ala Arg705 710 715 720Thr Gly Val Arg Arg Pro Ser Phe Ala Ser Ile
His Gly Arg Ser Ala 725 730 735Thr Glu Val His Ala Ser Asp Leu Thr
Leu Asp Lys Phe Ile Asp Ala 740 745 750Ala Thr Leu Ala Ala Ala Pro
Asn Leu Pro Ala Pro Ser Ala Gln Val 755 760 765Arg Thr Val Leu Leu
Thr Gly Ala Thr Gly Phe Leu Gly Arg Tyr Leu 770 775 780Ala Leu Glu
Trp Leu Asp Arg Met Asp Leu Val Asn Gly Lys Leu Ile785 790 795
800Cys Leu Val Arg Ala Arg Ser Asp Glu Glu Ala Gln Ala Arg Leu Asp
805 810 815Ala Thr Phe Asp Ser Gly Asp Pro Tyr Leu Val Arg His Tyr
Arg Glu 820 825 830Leu Gly Ala Gly Arg Leu Glu Val Leu Ala Gly
Asp
Lys Gly Glu Ala 835 840 845Asp Leu Gly Leu Asp Arg Val Thr Trp Gln
Arg Leu Ala Asp Thr Val 850 855 860Asp Leu Ile Val Asp Pro Ala Ala
Leu Val Asn His Val Leu Pro Tyr865 870 875 880Ser Gln Leu Phe Gly
Pro Asn Ala Ala Gly Thr Ala Glu Leu Leu Arg 885 890 895Leu Ala Leu
Thr Gly Lys Arg Lys Pro Tyr Ile Tyr Thr Ser Thr Ile 900 905 910Ala
Val Gly Glu Gln Ile Pro Pro Glu Ala Phe Thr Glu Asp Ala Asp 915 920
925Ile Arg Ala Ile Ser Pro Thr Arg Arg Ile Asp Asp Ser Tyr Ala Asn
930 935 940Gly Tyr Ala Asn Ser Lys Trp Ala Gly Glu Val Leu Leu Arg
Glu Ala945 950 955 960His Glu Gln Cys Gly Leu Pro Val Thr Val Phe
Arg Cys Asp Met Ile 965 970 975Leu Ala Asp Thr Ser Tyr Thr Gly Gln
Leu Asn Leu Pro Asp Met Phe 980 985 990Thr Arg Leu Met Leu Ser Leu
Ala Ala Thr Gly Ile Ala Pro Gly Ser 995 1000 1005Phe Tyr Glu Leu
Asp Ala His Gly Asn Arg Gln Arg Ala His Tyr 1010 1015 1020Asp Gly
Leu Pro Val Glu Phe Val Ala Glu Ala Ile Cys Thr Leu 1025 1030
1035Gly Thr His Ser Pro Asp Arg Phe Val Thr Tyr His Val Met Asn
1040 1045 1050Pro Tyr Asp Asp Gly Ile Gly Leu Asp Glu Phe Val Asp
Trp Leu 1055 1060 1065Asn Ser Pro Thr Ser Gly Ser Gly Cys Thr Ile
Gln Arg Ile Ala 1070 1075 1080Asp Tyr Gly Glu Trp Leu Gln Arg Phe
Glu Thr Ser Leu Arg Ala 1085 1090 1095Leu Pro Asp Arg Gln Arg His
Ala Ser Leu Leu Pro Leu Leu His 1100 1105 1110Asn Tyr Arg Glu Pro
Ala Lys Pro Ile Cys Gly Ser Ile Ala Pro 1115 1120 1125Thr Asp Gln
Phe Arg Ala Ala Val Gln Glu Ala Lys Ile Gly Pro 1130 1135 1140Asp
Lys Asp Ile Pro His Leu Thr Ala Ala Ile Ile Ala Lys Tyr 1145 1150
1155Ile Ser Asn Leu Arg Leu Leu Gly Leu Leu 1160
1165413507DNAMycobacterium smegmatis 41atgacgatcg aaacgcgcga
agaccgcttc aaccggcgca ttgaccactt gttcgaaacc 60gacccgcagt tcgccgccgc
ccgtcccgac gaggcgatca gcgcggctgc cgccgatccg 120gagttgcgcc
ttcctgccgc ggtcaaacag attctggccg gctatgcgga ccgccctgcg
180ctgggcaagc gcgccgtcga gttcgtcacc gacgaagaag gccgcaccac
cgcgaagctc 240ctgccccgct tcgacaccat cacctaccgt cagctcgcag
gccggatcca ggccgtgacc 300aatgcctggc acaaccatcc ggtgaatgcc
ggtgaccgcg tggccatcct gggtttcacc 360agtgtcgact acacgacgat
cgacatcgcc ctgctcgaac tcggcgccgt gtccgtaccg 420ctgcagacca
gtgcgccggt ggcccaactg cagccgatcg tcgccgagac cgagcccaag
480gtgatcgcgt cgagcgtcga cttcctcgcc gacgcagtcg ctctcgtcga
gtccgggccc 540gcgccgtcgc gactggtggt gttcgactac agccacgagg
tcgacgatca gcgtgaggcg 600ttcgaggcgg ccaagggcaa gctcgcaggc
accggcgtcg tcgtcgagac gatcaccgac 660gcactggacc gcgggcggtc
actcgccgac gcaccgctct acgtgcccga cgaggccgac 720ccgctgaccc
ttctcatcta cacctccggc agcaccggca ctcccaaggg cgcgatgtac
780cccgagtcca agaccgccac gatgtggcag gccgggtcca aggcccggtg
ggacgagacc 840ctcggcgtga tgccgtcgat caccctgaac ttcatgccca
tgagtcacgt catggggcgc 900ggcatcctgt gcagcacact cgccagcggc
ggaaccgcgt acttcgccgc acgcagcgac 960ctgtccacct tcctggagga
cctcgccctc gtgcggccca cgcagctcaa cttcgttcct 1020cgcatctggg
acatgctgtt ccaggagtac cagagccgcc tcgacaaccg ccgcgccgag
1080ggatccgagg accgagccga agccgcagtc ctcgaagagg tccgcaccca
actgctcggc 1140gggcgattcg tttcggccct gaccggatcg gctcccatct
cggcggagat gaagagctgg 1200gtcgaggacc tgctcgacat gcatctgctg
gagggctacg gctccaccga ggccggcgcg 1260gtgttcatcg acgggcagat
ccagcgcccg ccggtcatcg actacaagct ggtcgacgtg 1320cccgatctcg
gctacttcgc cacggaccgg ccctacccgc gcggcgaact tctggtcaag
1380tccgagcaga tgttccccgg ctactacaag cgtccggaga tcaccgccga
gatgttcgac 1440gaggacgggt actaccgcac cggcgacatc gtcgccgagc
tcgggcccga ccatctcgaa 1500tacctcgacc gccgcaacaa cgtgctgaaa
ctgtcgcagg gcgaattcgt cacggtctcc 1560aagctggagg cggtgttcgg
cgacagcccc ctggtacgcc agatctacgt ctacggcaac 1620agcgcgcggt
cctatctgct ggcggtcgtg gtcccgaccg aagaggcact gtcacgttgg
1680gacggtgacg aactcaagtc gcgcatcagc gactcactgc aggacgcggc
acgagccgcc 1740ggattgcagt cgtatgagat cccgcgtgac ttcctcgtcg
agacaacacc tttcacgctg 1800gagaacggcc tgctgaccgg tatccgcaag
ctggcccggc cgaaactgaa ggcgcactac 1860ggcgaacgcc tcgaacagct
ctacaccgac ctggccgagg ggcaggccaa cgagttgcgc 1920gagttgcgcc
gcaacggagc cgaccggccc gtggtcgaga ccgtcagccg cgccgcggtc
1980gcactgctcg gtgcctccgt cacggatctg cggtccgatg cgcacttcac
cgatctgggt 2040ggagattcgt tgtcggcctt gagcttctcg aacctgttgc
acgagatctt cgatgtcgac 2100gtgccggtcg gcgtcatcgt cagcccggcc
accgacctgg caggcgtcgc ggcctacatc 2160gagggcgaac tgcgcggctc
caagcgcccc acatacgcgt cggtgcacgg gcgcgacgcc 2220accgaggtgc
gcgcgcgtga tctcgccctg ggcaagttca tcgacgccaa gaccctgtcc
2280gccgcgccgg gtctgccgcg ttcgggcacc gagatccgca ccgtgctgct
gaccggcgcc 2340accgggttcc tgggccgcta tctggcgctg gaatggctgg
agcgcatgga cctggtggac 2400ggcaaggtga tctgcctggt gcgcgcccgc
agcgacgacg aggcccgggc gcgtctggac 2460gccacgttcg acaccgggga
cgcgacactg ctcgagcact accgcgcgct ggcagccgat 2520cacctcgagg
tgatcgccgg tgacaagggc gaggccgatc tgggtctcga ccacgacacg
2580tggcagcgac tggccgacac cgtcgatctg atcgtcgatc cggccgccct
ggtcaatcac 2640gtcctgccgt acagccagat gttcggaccc aatgcgctcg
gcaccgccga actcatccgg 2700atcgcgctga ccaccacgat caagccgtac
gtgtacgtct cgacgatcgg tgtgggacag 2760ggcatctccc ccgaggcgtt
cgtcgaggac gccgacatcc gcgagatcag cgcgacgcgc 2820cgggtcgacg
actcgtacgc caacggctac ggcaacagca agtgggccgg cgaggtcctg
2880ctgcgggagg cgcacgactg gtgtggtctg ccggtctcgg tgttccgctg
cgacatgatc 2940ctggccgaca cgacctactc gggtcagctg aacctgccgg
acatgttcac ccgcctgatg 3000ctgagcctcg tggcgaccgg catcgcgccc
ggttcgttct acgaactcga tgcggacggc 3060aaccggcagc gcgcccacta
cgacgggctg cccgtggagt tcatcgccga ggcgatctcc 3120accatcggct
cgcaggtcac cgacggattc gagacgttcc acgtgatgaa cccgtacgac
3180gacggcatcg gcctcgacga gtacgtggac tggctgatcg aggccggcta
ccccgtgcac 3240cgcgtcgacg actacgccac ctggctgagc cggttcgaaa
ccgcactgcg ggccctgccg 3300gaacggcaac gtcaggcctc gctgctgccg
ctgctgcaca actatcagca gccctcaccg 3360cccgtgtgcg gtgccatggc
acccaccgac cggttccgtg ccgcggtgca ggacgcgaag 3420atcggccccg
acaaggacat tccgcacgtc acggccgacg tgatcgtcaa gtacatcagc
3480aacctgcaga tgctcggatt gctgtaa 3507423507DNAMycobacterium
tuberculosis 42atgtcgatca acgatcagcg actgacacgc cgcgtcgagg
acctatacgc cagcgacgcc 60cagttcgccg ccgccagtcc caacgaggcg atcacccagg
cgatcgacca gcccggggtc 120gcgcttccac agctcatccg tatggtcatg
gagggctacg ccgatcggcc ggcactcggc 180cagcgtgcgc tccgcttcgt
caccgacccc gacagcggcc gcaccatggt cgagctactg 240ccgcggttcg
agaccatcac ctaccgcgaa ctgtgggccc gcgccggcac attggccacc
300gcgttgagcg ctgagcccgc gatccggccg ggcgaccggg tttgcgtgct
gggcttcaac 360agcgtcgact acacaaccat cgacatcgcg ctgatccggt
tgggcgccgt gtcggttcca 420ctgcagacca gtgcgccggt caccgggttg
cgcccgatcg tcaccgagac cgagccgacg 480atgatcgcca ccagcatcga
caatcttggc gacgccgtcg aagtgctggc cggtcacgcc 540ccggcccggc
tggtcgtatt cgattaccac ggcaaggttg acacccaccg cgaggccgtc
600gaagccgccc gagctcggtt ggccggctcg gtgaccatcg acacacttgc
cgaactgatc 660gaacgcggca gggcgctgcc ggccacaccc attgccgaca
gcgccgacga cgcgctggcg 720ctgctgattt acacctcggg tagtaccggc
gcacccaaag gcgccatgta tcgcgagagc 780caggtgatga gcttctggcg
caagtcgagt ggctggttcg agccgagcgg ttacccctcg 840atcacgctga
acttcatgcc gatgagccac gtcgggggcc gtcaggtgct ctacgggacg
900ctttccaacg gcggtaccgc ctacttcgtc gccaagagcg acctgtcgac
gctgttcgag 960gacctcgccc tggtgcggcc cacagaattg tgcttcgtgc
cgcgcatctg ggacatggtg 1020ttcgcagagt tccacagcga ggtcgaccgc
cgcttggtgg acggcgccga tcgagcggcg 1080ctggaagcgc aggtgaaggc
cgagctgcgg gagaacgtgc tcggcggacg gtttgtcatg 1140gcgctgaccg
gttccgcgcc gatctccgct gagatgacgg cgtgggtcga gtccctgctg
1200gccgacgtgc atttggtgga gggttacggc tccaccgagg ccgggatggt
cctgaacgac 1260ggcatggtgc ggcgccccgc ggtgatcgac tacaagctgg
tcgacgtgcc cgagctgggc 1320tacttcggca ccgatcagcc ctacccccgg
ggcgagctgc tggtcaagac gcaaaccatg 1380ttccccggct actaccagcg
cccggatgtc accgccgagg tgttcgaccc cgacggcttc 1440taccggaccg
gggacatcat ggccaaagta ggccccgacc agttcgtcta cctcgaccgc
1500cgcaacaacg tgctaaagct ctcccagggc gagttcatcg ccgtgtcgaa
gctcgaggcg 1560gtgttcggcg acagcccgct ggtccgacag atcttcatct
acggcaacag tgcccgggcc 1620tacccgctgg cggtggttgt cccgtccggg
gacgcgcttt ctcgccatgg catcgagaat 1680ctcaagcccg tgatcagcga
gtccctgcag gaggtagcga gggcggccgg cctgcaatcc 1740tacgagattc
cacgcgactt catcatcgaa accacgccgt tcaccctgga gaacggcctg
1800ctcaccggca tccgcaagct ggcacgcccg cagttgaaga agttctatgg
cgaacgtctc 1860gagcggctct ataccgagct ggccgatagc caatccaacg
agctgcgcga gctgcggcaa 1920agcggtcccg atgcgccggt gcttccgacg
ctgtgccgtg ccgcggctgc gttgctgggc 1980tctaccgctg cggatgtgcg
gccggacgcg cacttcgccg acctgggtgg tgactcgctc 2040tcggcgctgt
cgttggccaa cctgctgcac gagatcttcg gcgtcgacgt gccggtgggt
2100gtcattgtca gcccggcaag cgacctgcgg gccctggccg accacatcga
agcagcgcgc 2160accggcgtca ggcgacccag cttcgcctcg atacacggtc
gctccgcgac ggaagtgcac 2220gccagcgacc tcacgctgga caagttcatc
gacgctgcca ccctggccgc agccccgaac 2280ctgccggcac cgagcgccca
agtgcgcacc gtactgctga ccggcgccac cggctttttg 2340ggtcgctacc
tggcgctgga atggctcgac cgcatggacc tggtcaacgg caagctgatc
2400tgcctggtcc gcgccagatc cgacgaggaa gcacaagccc ggctggacgc
gacgttcgat 2460agcggcgacc cgtatttggt gcggcactac cgcgaattgg
gcgccggccg cctcgaggtg 2520ctcgccggcg acaagggcga ggccgacctg
ggcctggacc gggtcacctg gcagcggcta 2580gccgacacgg tggacctgat
cgtggacccc gcggccctgg tcaaccacgt gctgccgtat 2640agccagctgt
tcggcccaaa cgcggcgggc accgccgagt tgcttcggct ggcgctgacc
2700ggcaagcgca agccatacat ctacacctcg acgatcgccg tgggcgagca
gatcccgccg 2760gaggcgttca ccgaggacgc cgacatccgg gccatcagcc
cgacccgcag gatcgacgac 2820agctacgcca acggctacgc gaacagcaag
tgggccggcg aggtgctgct gcgcgaagct 2880cacgagcagt gcggcctgcc
ggtgacggtc ttccgctgcg acatgatcct ggccgacacc 2940agctataccg
gtcagctcaa cctgccggac atgttcaccc ggctgatgct gagcctggcc
3000gctaccggca tcgcacccgg ttcgttctat gagctggatg cgcacggcaa
tcggcaacgc 3060gcccactatg acggcttgcc ggtcgaattc gtcgcagaag
ccatttgcac ccttgggaca 3120catagcccgg accgttttgt cacctaccac
gtgatgaacc cctacgacga cggcatcggg 3180ctggacgagt tcgtcgactg
gctcaactcc ccaactagcg ggtccggttg cacgatccag 3240cggatcgccg
actacggcga gtggctgcag cggttcgaga cttcgctgcg tgccttgccg
3300gatcgccagc gccacgcctc gctgctgccc ttgctgcaca actaccgaga
gcctgcaaag 3360ccgatatgcg ggtcaatcgc gcccaccgac cagttccgcg
ctgccgtcca agaagcgaaa 3420atcggtccgg acaaagacat tccgcacctc
acggcggcga tcatcgcgaa gtacatcagc 3480aacctgcgac tgctcgggct gctgtga
3507433522DNAMycobacterium smegmatis 43atgaccagcg atgttcacga
cgccacagac ggcgtcaccg aaaccgcact cgacgacgag 60cagtcgaccc gccgcatcgc
cgagctgtac gccaccgatc ccgagttcgc cgccgccgca 120ccgttgcccg
ccgtggtcga cgcggcgcac aaacccgggc tgcggctggc agagatcctg
180cagaccctgt tcaccggcta cggtgaccgc ccggcgctgg gataccgcgc
ccgtgaactg 240gccaccgacg agggcgggcg caccgtgacg cgtctgctgc
cgcggttcga caccctcacc 300tacgcccagg tgtggtcgcg cgtgcaagcg
gtcgccgcgg ccctgcgcca caacttcgcg 360cagccgatct accccggcga
cgccgtcgcg acgatcggtt tcgcgagtcc cgattacctg 420acgctggatc
tcgtatgcgc ctacctgggc ctcgtgagtg ttccgctgca gcacaacgca
480ccggtcagcc ggctcgcccc gatcctggcc gaggtcgaac cgcggatcct
caccgtgagc 540gccgaatacc tcgacctcgc agtcgaatcc gtgcgggacg
tcaactcggt gtcgcagctc 600gtggtgttcg accatcaccc cgaggtcgac
gaccaccgcg acgcactggc ccgcgcgcgt 660gaacaactcg ccggcaaggg
catcgccgtc accaccctgg acgcgatcgc cgacgagggc 720gccgggctgc
cggccgaacc gatctacacc gccgaccatg atcagcgcct cgcgatgatc
780ctgtacacct cgggttccac cggcgcaccc aagggtgcga tgtacaccga
ggcgatggtg 840gcgcggctgt ggaccatgtc gttcatcacg ggtgacccca
cgccggtcat caacgtcaac 900ttcatgccgc tcaaccacct gggcgggcgc
atccccattt ccaccgccgt gcagaacggt 960ggaaccagtt acttcgtacc
ggaatccgac atgtccacgc tgttcgagga tctcgcgctg 1020gtgcgcccga
ccgaactcgg cctggttccg cgcgtcgccg acatgctcta ccagcaccac
1080ctcgccaccg tcgaccgcct ggtcacgcag ggcgccgacg aactgaccgc
cgagaagcag 1140gccggtgccg aactgcgtga gcaggtgctc ggcggacgcg
tgatcaccgg attcgtcagc 1200accgcaccgc tggccgcgga gatgagggcg
ttcctcgaca tcaccctggg cgcacacatc 1260gtcgacggct acgggctcac
cgagaccggc gccgtgacac gcgacggtgt gatcgtgcgg 1320ccaccggtga
tcgactacaa gctgatcgac gttcccgaac tcggctactt cagcaccgac
1380aagccctacc cgcgtggcga actgctggtc aggtcgcaaa cgctgactcc
cgggtactac 1440aagcgccccg aggtcaccgc gagcgtcttc gaccgggacg
gctactacca caccggcgac 1500gtcatggccg agaccgcacc cgaccacctg
gtgtacgtgg accgtcgcaa caacgtcctc 1560aaactcgcgc agggcgagtt
cgtggcggtc gccaacctgg aggcggtgtt ctccggcgcg 1620gcgctggtgc
gccagatctt cgtgtacggc aacagcgagc gcagtttcct tctggccgtg
1680gtggtcccga cgccggaggc gctcgagcag tacgatccgg ccgcgctcaa
ggccgcgctg 1740gccgactcgc tgcagcgcac cgcacgcgac gccgaactgc
aatcctacga ggtgccggcc 1800gatttcatcg tcgagaccga gccgttcagc
gccgccaacg ggctgctgtc gggtgtcgga 1860aaactgctgc ggcccaacct
caaagaccgc tacgggcagc gcctggagca gatgtacgcc 1920gatatcgcgg
ccacgcaggc caaccagttg cgcgaactgc ggcgcgcggc cgccacacaa
1980ccggtgatcg acaccctcac ccaggccgct gccacgatcc tcggcaccgg
gagcgaggtg 2040gcatccgacg cccacttcac cgacctgggc ggggattccc
tgtcggcgct gacactttcg 2100aacctgctga gcgatttctt cggtttcgaa
gttcccgtcg gcaccatcgt gaacccggcc 2160accaacctcg cccaactcgc
ccagcacatc gaggcgcagc gcaccgcggg tgaccgcagg 2220ccgagtttca
ccaccgtgca cggcgcggac gccaccgaga tccgggcgag tgagctgacc
2280ctggacaagt tcatcgacgc cgaaacgctc cgggccgcac cgggtctgcc
caaggtcacc 2340accgagccac ggacggtgtt gctctcgggc gccaacggct
ggctgggccg gttcctcacg 2400ttgcagtggc tggaacgcct ggcacctgtc
ggcggcaccc tcatcacgat cgtgcggggc 2460cgcgacgacg ccgcggcccg
cgcacggctg acccaggcct acgacaccga tcccgagttg 2520tcccgccgct
tcgccgagct ggccgaccgc cacctgcggg tggtcgccgg tgacatcggc
2580gacccgaatc tgggcctcac acccgagatc tggcaccggc tcgccgccga
ggtcgacctg 2640gtggtgcatc cggcagcgct ggtcaaccac gtgctcccct
accggcagct gttcggcccc 2700aacgtcgtgg gcacggccga ggtgatcaag
ctggccctca ccgaacggat caagcccgtc 2760acgtacctgt ccaccgtgtc
ggtggccatg gggatccccg acttcgagga ggacggcgac 2820atccggaccg
tgagcccggt gcgcccgctc gacggcggat acgccaacgg ctacggcaac
2880agcaagtggg ccggcgaggt gctgctgcgg gaggcccacg atctgtgcgg
gctgcccgtg 2940gcgacgttcc gctcggacat gatcctggcg catccgcgct
accgcggtca ggtcaacgtg 3000ccagacatgt tcacgcgact cctgttgagc
ctcttgatca ccggcgtcgc gccgcggtcg 3060ttctacatcg gagacggtga
gcgcccgcgg gcgcactacc ccggcctgac ggtcgatttc 3120gtggccgagg
cggtcacgac gctcggcgcg cagcagcgcg agggatacgt gtcctacgac
3180gtgatgaacc cgcacgacga cgggatctcc ctggatgtgt tcgtggactg
gctgatccgg 3240gcgggccatc cgatcgaccg ggtcgacgac tacgacgact
gggtgcgtcg gttcgagacc 3300gcgttgaccg cgcttcccga gaagcgccgc
gcacagaccg tactgccgct gctgcacgcg 3360ttccgcgctc cgcaggcacc
gttgcgcggc gcacccgaac ccacggaggt gttccacgcc 3420gcggtgcgca
ccgcgaaggt gggcccggga gacatcccgc acctcgacga ggcgctgatc
3480gacaagtaca tacgcgatct gcgtgagttc ggtctgatct ga
3522443582DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 44atgggcagca
gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60atgacgagcg
atgttcacga cgcgaccgac ggcgttaccg agactgcact ggatgatgag
120cagagcactc gtcgtattgc agaactgtac gcaacggacc cagagttcgc
agcagcagct 180cctctgccgg ccgttgtcga tgcggcgcac aaaccgggcc
tgcgtctggc ggaaatcctg 240cagaccctgt tcaccggcta cggcgatcgt
ccggcgctgg gctatcgtgc acgtgagctg 300gcgacggacg aaggcggtcg
tacggtcacg cgtctgctgc cgcgcttcga taccctgacc 360tatgcacagg
tgtggagccg tgttcaagca gtggctgcag cgttgcgtca caatttcgca
420caaccgattt acccgggcga cgcggtcgcg actatcggct ttgcgagccc
ggactatttg 480acgctggatc tggtgtgcgc gtatctgggc ctggtcagcg
ttcctttgca gcataacgct 540ccggtgtctc gcctggcccc gattctggcc
gaggtggaac cgcgtattct gacggtgagc 600gcagaatacc tggacctggc
ggttgaatcc gtccgtgatg tgaactccgt cagccagctg 660gttgttttcg
accatcatcc ggaagtggac gatcaccgtg acgcactggc tcgcgcacgc
720gagcagctgg ccggcaaagg tatcgcagtt acgaccctgg atgcgatcgc
agacgaaggc 780gcaggtttgc cggctgagcc gatttacacg gcggatcacg
atcagcgtct ggccatgatt 840ctgtatacca gcggctctac gggtgctccg
aaaggcgcga tgtacaccga agcgatggtg 900gctcgcctgt ggactatgag
ctttatcacg ggcgacccga ccccggttat caacgtgaac 960ttcatgccgc
tgaaccatct gggcggtcgt atcccgatta gcaccgccgt gcagaatggc
1020ggtaccagct acttcgttcc ggaaagcgac atgagcacgc tgtttgagga
tctggccctg 1080gtccgcccta ccgaactggg tctggtgccg cgtgttgcgg
acatgctgta ccagcatcat 1140ctggcgaccg tggatcgcct ggtgacccag
ggcgcggacg aactgactgc ggaaaagcag 1200gccggtgcgg aactgcgtga
acaggtcttg ggcggtcgtg ttatcaccgg ttttgtttcc 1260accgcgccgt
tggcggcaga gatgcgtgct tttctggata tcaccttggg tgcacacatc
1320gttgacggtt acggtctgac cgaaaccggt gcggtcaccc gtgatggtgt
gattgttcgt 1380cctccggtca ttgattacaa gctgatcgat gtgccggagc
tgggttactt ctccaccgac 1440aaaccgtacc cgcgtggcga gctgctggtt
cgtagccaaa cgttgactcc gggttactac 1500aagcgcccag aagtcaccgc
gtccgttttc gatcgcgacg gctattacca caccggcgac 1560gtgatggcag
aaaccgcgcc agaccacctg gtgtatgtgg accgccgcaa caatgttctg
1620aagctggcgc aaggtgaatt tgtcgccgtg gctaacctgg aggccgtttt
cagcggcgct 1680gctctggtcc gccagatttt cgtgtatggt aacagcgagc
gcagctttct gttggctgtt 1740gttgtcccta ccccggaggc gctggagcaa
tacgaccctg ccgcattgaa agcagccctg 1800gcggattcgc tgcagcgtac
ggcgcgtgat gccgagctgc agagctatga agtgccggcg 1860gacttcattg
ttgagactga gccttttagc gctgcgaacg gtctgctgag cggtgttggc
1920aagttgctgc gtccgaattt gaaggatcgc tacggtcagc gtttggagca
gatgtacgcg 1980gacatcgcgg ctacgcaggc gaaccaattg cgtgaactgc
gccgtgctgc ggctactcaa 2040ccggtgatcg acacgctgac gcaagctgcg
gcgaccatcc tgggtaccgg cagcgaggtt 2100gcaagcgacg cacactttac
tgatttgggc ggtgattctc tgagcgcgct gacgttgagc 2160aacttgctgt
ctgacttctt tggctttgaa gtcccggttg gcacgattgt taacccagcg
2220actaatctgg
cacagctggc gcaacatatc gaggcgcagc gcacggcggg tgaccgccgt
2280ccatccttta cgacggtcca cggtgcggat gctacggaaa tccgtgcaag
cgaactgact 2340ctggacaaat tcatcgacgc tgagactctg cgcgcagcac
ctggtttgcc gaaggttacg 2400actgagccgc gtacggtcct gttgagcggt
gccaatggtt ggttgggccg cttcctgacc 2460ctgcagtggc tggaacgttt
ggcaccggtt ggcggtaccc tgatcaccat tgtgcgcggt 2520cgtgacgatg
cagcggcacg tgcacgtttg actcaggctt acgatacgga cccagagctg
2580tcccgccgct tcgctgagtt ggcggatcgc cacttgcgtg tggtggcagg
tgatatcggc 2640gatccgaatc tgggcctgac cccggagatt tggcaccgtc
tggcagcaga ggtcgatctg 2700gtcgttcatc cagcggccct ggtcaaccac
gtcctgccgt accgccagct gtttggtccg 2760aatgttgttg gcaccgccga
agttatcaag ttggctctga ccgagcgcat caagcctgtt 2820acctacctgt
ccacggttag cgtcgcgatg ggtattcctg attttgagga ggacggtgac
2880attcgtaccg tcagcccggt tcgtccgctg gatggtggct atgcaaatgg
ctatggcaac 2940agcaagtggg ctggcgaggt gctgctgcgc gaggcacatg
acctgtgtgg cctgccggtt 3000gcgacgtttc gtagcgacat gattctggcc
cacccgcgct accgtggcca agtgaatgtg 3060ccggacatgt tcacccgtct
gctgctgtcc ctgctgatca cgggtgtggc accgcgttcc 3120ttctacattg
gtgatggcga gcgtccgcgt gcacactacc cgggcctgac cgtcgatttt
3180gttgcggaag cggttactac cctgggtgct cagcaacgtg agggttatgt
ctcgtatgac 3240gttatgaatc cgcacgatga cggtattagc ttggatgtct
ttgtggactg gctgattcgt 3300gcgggccacc caattgaccg tgttgacgac
tatgatgact gggtgcgtcg ttttgaaacc 3360gcgttgaccg ccttgccgga
gaaacgtcgt gcgcagaccg ttctgccgct gctgcatgcc 3420tttcgcgcgc
cacaggcgcc gttgcgtggc gcccctgaac cgaccgaagt gtttcatgca
3480gcggtgcgta ccgctaaagt cggtccgggt gatattccgc acctggatga
agccctgatc 3540gacaagtaca tccgtgacct gcgcgagttc ggtctgattt ag
35824566DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 45cggttctggc aaatattctg
aaatgagctg ttgacaatta atcaaatccg gctcgtataa 60tgtgtg
664630DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 46ggtttattcc tccttattta atcgatacat
304759DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 47atgtatcgat taaataagga ggaataaacc
atgggcacga gcgatgttca cgacgcgac 594859DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 48atgtatcgat taaataagga ggaataaacc gtgggcacga gcgatgttca
cgacgcgac 594959DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 49atgtatcgat taaataagga
ggaataaacc ttgggcacga gcgatgttca cgacgcgac 595026DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 50ttctaaatca gaccgaactc gcgcag 265148DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 51ctgcgcgagt tcggtctgat ttagaattcc tcgaggatgg tagtgtgg
485227DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 52cagtcgacat acgaaacggg aatgcgg
275356DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 53ccgcattccc gtttcgtatg tcgactgaaa
cctcaggcat tgagaagcac acggtc 565456DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
primer" 54ctcatttcag aatatttgcc agaaccgtta atttcctaat gcaggagtcg
cataag 565520DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic primer" 55ggatctcgac gctctccctt
205628DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic primer" 56tcaaaaacgc cattaacctg atgttctg 28
* * * * *